WO2004069218A1 - Use of verbascoside as a stimulant agent for the production of thermal shock proteins by the cells of the skin - Google Patents

Abstract

The invention relates to the cosmetic use of a phenyl propanoid glycoside, verbascoside, as an agent for the stimulation of the production of thermal shock proteins and also for the inhibition of collagenase and elastase and stimulation of hydroxylases, by the cells of the skin.

Description

 USE OF VERBASCOSIDE AS A STIMULATING AGENT FOR THE SYNTHESIS OF THERMAL SHOCK PROTEINS BY SKIN CELLS.

The present invention relates to the field of cosmetic compositions for combating aging and aggressions of the skin. More particularly the invention relates to the cosmetic use of a phenylpropanoid glycoside, the verbascoside, as an agent on the one hand for stimulating the synthesis of heat shock proteins, and on the other hand for inhibiting collagenase and elastase and stimulation of hydroxylases, by skin cells.

The term “phenylpropanoid glycoside” denotes a group of natural products frequently present in plants of the order Tubiflorales.

The phenyl propanoid glycosides are obtained by joining 3 basic units: - a central glucose, a C ₆ -C _{2 group} , often it is dihydroxyphenyl-β-ethanol linked to the hemiacetal function of glucose,

- A C ₆ -C _{3 group} , often it is hydroxycinnamic acid, in acyl on one of the remaining OH functions of glucose.

The aromatic unit can be substituted differently. Hydroxycinnamic acid is then replaced by caffeic acid for example, and other saccharides are often linked to 1 or 2 of the free hydroxyls of central glucose.

The first phenylpropanoid glycoside extracted from Verbascum sinatum was described in 1963 under the name of verbascoside. Its complete structure was elucidated in 1968, under the name actéoside. The similarity between these two entities is now established, and we will use in the context of the present invention the original designation of verbascoside. Verbascoside, whose formula is shown below, has a rhamnose residue on the carbon 3 of glucose. Its full chemical name is then _- (3 ', 4'- dihydroxyphenyl) ethyl-O-α-L-rhamnopyranosyl- (l-> 3) - -D- (4-

0-caffeoyl) -glucopyranoside:

Verbascoside = actéoside R1=R3=H ; R2=Rha

Verbascoside = actoside R1 = R3 = H; R2 = Rha

The phenyl propanoid glycosides are presented by

Khalil Taoubi (1992, doctoral thesis, National Institute

Polytechnique de Toulouse) as heteroside esters of caffeic acid (EHAC). These molecules are studied in the genera of the family Verbenaceae.

The chemotaxonomic distribution of verbascoside is much wider, essentially in the order of Tubifloreae in almost all families, for example:

Acanthaceae (Acanthus sp, Crossandra sp.), Asteraceae

(Echinacea sp.), Bignoniaceae (Campsis sp., Paulownia sp.),

Buddlejaceae (Buddleja sp.), Gesneriaceae (Conandron sp.),

La iaceae (Ajuga sp., Leucoseptrum s., Stachys sp.), Oleaceae (Forsythia sp., Fraxinus sp., Syringa s.),

Orobanchaceae (Cistanche sp., Orobanche sp.), Pedaliaceae

(Harpagophytum sp., Sesamum sp.), Plantaginaceae (Plantago sp.), Scrophulariaceae (Calceolaria sp., Pedicularis sp. Rhemannia sp., Scrophularia s., Verbascum sp.), Verbenaceae (Clerodendron sp., Lantana sp., Lippia sp., Verbena sp.).

Phenylpropanoid glycosides are known in the prior art to exhibit therapeutic activities in anti-fungal, anti-bacterial, anti-viral, analgesic applications. Khalil Taoubi's thesis mentioned above studies the role of verbascoside on the inhibition of protein kinase C. This enzyme is involved in the mechanisms of cellular signal transmissions, and PKC inhibitors have a powerful anti-mitotic activity, allowing to fight against tumor proliferation, tumor angiogenesis, atherosclerosis.

More recently, properties of verbascoside have been demonstrated to treat dermatological problems. US Patent 6171593 describes the use of mullein (V. thapsus) for the treatment of skin disorders: actinic keratoses, seborrheic keratoses, prurigo, psoriasis, contact dermatitis.

In the cosmetic field, Japanese patent JP 11171723 relates to the application of an extract of Verbascum thapsus as an antioxidant agent to combat the oxidation of the lipids of the skin and therefore its aging.

The research carried out in the context of the invention has now enabled the Applicant to highlight surprising properties of verbascoside, making it possible to use it in the field of cosmetics, in particular for combating aging of the skin. The invention relates to everything particularly the use of a phenyl propanoid glycoside, such as the verbascoside, for the manufacture of a cosmetic or dermatological composition to combat the decrease in integrity of the dermis and also against the aggressions of the skin by the environment, such as temperature, pollution, UV, etc.

In particular, the Applicant has now shown that verbascoside is capable of stimulating the synthesis of heat shock proteins, and more particularly of HSP 70, by the skin cells and thus of acting effectively against the aggressions of these by the environment.

HSPs represent a family of highly conserved proteins, naturally present in all cells of living organisms (humans, animals, plants, bacteria). They are synthesized in response to an increase in temperature (hence the first name of Heat Shock proteins (HSPs)) but also in response to other stress promoters of protein denaturation such as heavy metals (cadmium), ethanol, amino acid analogs or metabolic poisons (TBT: tributyltin).

In general, HSPs would recognize hydrophobic regions normally buried within the protein, but which become accessible in the case of proteins that are not yet folded or denatured. HSPs are able to control the folding of other proteins, called chaperone function, under normal conditions, in the absence of any stressor. After exposure to a stressor, HSPs prevent or correct the denaturation and aggregation of proteins. If the protein is very denatured, the chaperone action may give way to a protease action: when the protein is irreparable, it is degraded and eliminated.

Since the human organism is an open system, it is forced to interact with its environment. Each interaction is summed up by a breakdown in cellular homeostasis (or stress), by a search for a new balance and by a general adaptive response.

During aging, the adaptive response to stress is greatly altered. In old cells, basic expression and induction of HSP are less effective than in young cells.

Nearly thirty HSPs have been discovered in recent years. These proteins are classified according to their molecular weight. There are thus four families: the HSP 90, the HSP 70, the HSP 60, and the small HSPs. Thus, in the HSP 70 family, HSPs of molecular weight close to 70 kDa will be found.

Schematically, we can group HSPs into 2 families: constitutive HSPs and inducible HSPs. The constituent HSPs are responsible for the conformation of newly synthesized proteins, the transport of proteins and the activation of their function. The synthesis of constitutive HSP evolves with age, and generally tends to decrease in the tissues. There is an interest here in stimulating the basic expression of the constitutive HSPs in order to combat a negative development (see result of the induction of HSPs 70 by the verbascoside on cells in the absence of stress).

Inducible HSPs, as their name suggests, see their rate increase after stress. However, with age, it can be seen that the response time to stress increases. HSPs have four main roles:

- as soon as they leave the ribosomes, they ensure the proper folding of the protein into a functional structure

- they "correct" proteins which, for one reason or another (mutation, denaturation) have undergone degradation

- they assist the proteins migrating to another compartment while maintaining their unrolled structure without alteration, the time of passage through the membrane - they ensure the elimination of proteins whose errors are irreversible (actions of proteases or chaperones towards the organelles of degradation ).

The most studied heat shock proteins are HSC70 and HSP 70. HSC70 represents the constitutive protein with a molecular weight close to 70 kDa. HSP 70 is the inducible protein. HSP 70 is expressed at a low level in non-stressed cells. Increasing the expression HSP 70 can be seen as a means of increasing the fight against environmental pollution.

Thus, the invention relates to the cosmetic use of a phenyl propanoid glycoside, more particularly of verbascoside, as an agent stimulating the synthesis of a heat shock protein, more particularly HSP 70, by skin cells, said agent being incorporated into a cosmetic composition comprising one or more acceptable excipients.

The work carried out within the framework of the invention has also enabled the Applicant to highlight advantageous properties of verbascoside for combating the aging of the skin linked to the deterioration of the maintenance of the dermis. Aging of the dermis is the alteration that causes the most visible changes in the skin. It depends on two types of mechanisms, the first being the reduction in the mass of macromolecules that make up the connective tissue and the second a change in the architectural organization of the polymers that ensure the mechanical properties of the dermis. The quantity of macromolecules constituting the connective (collagens, elastins) is the result of protein synthesis of neoformation of these molecules and the degradation of these polymers by specific enzymes, collagenases and elastases. During the aging of the skin, the balance changes, syntheses slow down in favor of the increase in degradations. The Applicant has now shown that the verbascoside could oppose these phenomena, and therefore fight against the degradation of the dermis, therefore the aging of the skin.

One of the desired effects is to fight against the enzymes which degrade the support fibers of the dermis, but also to stimulate the maturation of collagen by the ibroblasts, in order to increase the quantity of macromolecules in the connective tissue.

It has thus been demonstrated the effectiveness of verbascoside in inhibiting collagenase by an in vitro model. The activity of collagenase is determined by hydrolysis of a specific and water-soluble substrate, representative of collagen, which is naturally insoluble. The substrate is split into two parts, releasing a residue, the absorption kinetics of which are monitored at 320 nm. The experiment is validated by the use of a reference witness.

The use of verbascoside in the reaction medium effectively reduces the activity of collagenase, thus preserving the substrate representative of the amount of collagen thus protected.

Similarly, the Applicant has studied the ability of verbascoside to inhibit the activity of elastase on an in vitro model. The activity of the elastase is determined on a specific and water-soluble substrate. The substrate reacts with the enzyme to release the p. Nitroalinine which absorbs at 386 nm. The activity of the enzyme is defined relative to the slope of the release kinetics of p. Nitroalinine.

The use of verbascoside in the reaction medium considerably reduces the slope of the kinetics of appearance of the marker, witnessing a measured efficiency of inhibition of the activity of the enzyme. Another desired effect is to ensure the good quality of the newly formed collagen, so as to restore a satisfactory architectural organization. The ability of the verbascoside to increase the maturation of collagen can be investigated by studying the extracellular matrix produced by fibroblasts in monolayer cultures exposed to the verbascoside.

Finally, it is known that collagen undergoes maturation stages at the time of its synthesis then of its secretion in the extracellular medium, then during its assembly in triple helix. One of these maturations is the hydroxylation of the proline and lysine residues by cellular enzymes, respectively prolyl hydroxylase and lysyl hydroxylase. Vitamin C (ascorbic acid) plays a fundamental role here by degrading the Fe ³⁺ -0 ^" complex formed during reactions, which inhibits hydroxylases.

The invention also relates to the use of verbascoside on this maturation step, so as to obtain an effective property on the quality of the collagen synthesized in the dermis. In view of all these results, anti-collagenase activity and anti-elastase activity demonstrated, activity of stimulating collagen synthesis and activity of collagen maturation, the invention proposes to use verbascoside to combat the decrease in the quantity and the quality of the connective tissue support fibers.

Thus, the invention relates to the use of a phenylpropanoid glycoside, more particularly of verbascoside, as an agent inhibiting collagenase and elastase and stimulating the maturation of collagen by an action on hydroxylase for the preparation of a cosmetic or dermatological composition intended to combat the decrease in the integrity of the dermis linked to the degradation of the skin's support fibers, which are collagen and elastin.

The phenylpropanoid glycoside, more particularly the verbascoside, used in the context of the invention can be in the form of a pure molecule, obtained by chemical synthesis, by enzymatic synthesis, or obtained by extraction and purification from fresh or dried plants. According to an advantageous embodiment of the invention, it can also be a plant extract rich in phenyl propanoid glycoside, more particularly in verbascoside. Such an extract of plant material has a concentration of phenylpropanoid glycoside, more particularly of verbascoside of between approximately 0.01 to 75%, preferably between 0.1 and 50%.

In all cases, it can be used as a free molecule, or encapsulated, vectorized, grafted, in order for example to improve its stability or its solubility, or even to reduce the color impact linked to its use. In the case of more or less extensive extraction from plants, the organs used can be the roots and / or the leaves and / or flowering tops or the whole plant. Verbascoside can also be obtained from plant cells in in vitro cultures, in suspension or in callus.

The plants used to obtain phenyl propanoid glycoside, more particularly verbascoside are preferably chosen, without being limiting, from the order of Tubiflorales.

More specifically, we will choose plants available on a large scale to meet industrial needs, from the genera Verbascum, Plantago, Verbena, Lippia, Fraxinus. The main representative of the genus Verbascum is white broth. Under the name white broth, we mean two officinal species which are V. densiflorum Bertol. (syn V. thapsiforme Schrod.) and V. phlomoïdes (from the Scrophulariaceae family). We can also cite mullein (V. thapsus).

In the genus Plantago, 3 species of plantains can be used: P. major L., P. media L., P. lanceolata L. (family Plantaginaceae). We use the whole plant or the leaves. The verbascoside content is approximately 0.2 to 4%.

In Verbenaceae, verbena (Lippia ci triodora = Aloysia tryphilla) and verbena officinalis

(VerJbeπa officinalis) can be used. All parts of the plant contain verbascoside, with an average content of around 5%.

For ash (Fraxinus ornus and F. excelsior - family Oleaceae), the bark and leaves contain verbascoside. Even if the verbascoside according to the present invention can come from these two parts of the plant, we preferentially choose leaves, renewable and more ecological resources, which have an average content of 4% of verbascoside.

Phenylpropanoid glycosides, and in particular verbascoside, are extracted from plants, usually using organic solvents (methanol, and to a lesser extent, ethanol or ethyl acetate), followed by dissolution of the crude extract in water, possibly hot, and washing with organic solvents. An optional step, but which improves the process, consists in treating the water-soluble fraction, either by freeze-drying it, or by extracting it with butanol, before chromatography. The use of silica gel and chloroform - methanol or chloroform - methanol - water is very frequent. Gentle methods (Sephadex LH-20 - Amersham Biosciences) that separate glycosidic and non-glycosidic compounds are recommended.

Depending on the desired degree of purity, one can just take up the methanolic extract put back to dryness in a suitable glycol-water solvent, or chain the purification steps until obtaining the pure molecule, or carry out any preparation which leads to an intermediate purity. .

For example, according to a particular embodiment, the verbascoside is purified by:

- grinding the dried plant (for verbena, it is advantageous to remove the essential oil by washing with petroleum ether before extraction) and extraction with stirring with methanol or methanol - water (80-20), at room temperature or hot at reflux;

- Concentration of the alcoholic solution obtained, under vacuum, and addition of water.

Washing can be done in 2 ways: - Washing with ethyl acetate and putting the fraction thus extracted to dryness. This fraction is then chromatographed in reverse phase and eluted with a 0 to 10% ethanol gradient. A fraction containing the pure verbascoside is obtained, which can be put back to dryness.

- Wash with n-butanol - ethyl acetate (5: 1) and put back to dryness. Chromatography is then carried out on Sephadex LH-20, with a 0-20% gradient of ethanol in water. A fraction containing the pure verbascoside is obtained, which can be put back to dryness.

For example, a particular preparation process includes the following steps:

- grinding the dried plant (for verbena, it is advantageous to remove the essential oil by washing with petroleum ether before extraction) and extraction with stirring with methanol or methanol - water (80-20), at room temperature or hot at reflux;

- Concentration of the alcoholic solution obtained, under vacuum, and taken up in water; - Extraction with ethyl acetate, then extraction with n-butanol which is concentrated under vacuum;

- a first chromatography is carried out on silica, eluted with a chloroform - methanol - water mixture (6: 4: 1); - a second chromatography is carried out on silica, eluted with a chloroform - methanol - water mixture (7: 3: 0.5);

- then passage over Sephadex LH-20 with a methanol-water solvent (1: 1) leads to a fraction containing the pure verbascoside.

Advantageously, the phenyl propanoid glycoside, more particularly the verbascoside, is present in a cosmetic composition according to the invention. a concentration of the order of 0.01 to 5%, preferably 0.05 to 2%, by weight of the composition.

The compositions according to the invention can be in any form and formulation known to a person skilled in the art.

Other advantages and characteristics of the invention will appear from the following examples relating to the properties of verbascoside and to the characteristics of compositions containing it.

Example 1: Induction of HSP by verbascoside.

This study highlighted the effect of verbascoside on the induction of stress genes in vitro. The tests were carried out on cells in cultures: young and old IMR90 human fibroblasts.

Studies showing the effects of aging on the expression of HSP 70 in fibroblasts have mainly been carried out on human pulmonary fibroblasts aged in vitro: IMR90, WI38 and MRC5.

Work by Conrad et al. (Exp Cell Re, 1998. 241 (2): p.404-413) made it possible to validate the IMR90 model, by observing similar effects of aging on HSP70 (decrease in RNA and protein) in IMR90 aged in vitro and on skin fibroblasts of young and old subjects.

From young or old cells, with or without the presence of stress (heavy and thermal metals), the induction of HSP70 mRNA by the verbascoside present in the culture medium is measured. After treatment to create stress and incubation, the cells are lysed and the mRNAs analyzed by Northern blot. Observation of cells in the absence of stress shows that the treatment with verbascoside slightly induces the mRNA of HSP70, as much for short exposures (6 h) as long exposures (3 times in 7 days). This property, of low intensity under these conditions, makes it possible to place the cell in an environment with a slightly higher cytosolic content in HSP, and therefore to protect the cells in prevention of possible stress. When stress is applied with the aid of cadmium to cells in cultures, it is observed that in the presence of verbascoside, the mRNAs of HSP70 are increased very significantly, in young and old cells. Verbascoside therefore helps to combat the environmental stress generated by heavy metals.

In the presence of a thermal shock, the verbascoside also induces the mRNA of there HSP70, specifically in the young cells.

In order to verify the induction of HSP 70 by the verbascoside on cells subjected to stress or not, cells (old or young) are seeded in flasks with complete MEM medium + 5% FCS, cultured up to 50 % of confluence. The day before the stimulation, the medium is replaced by MEM + 2% of FCS, then again replaced by medium comprising 1% of FCS for the experiment.

Heavy metal stress is achieved with cadmium at 10 μM. The cells are incubated for 6 h at 37 ° C., then lysed.

The RNAs are extracted with a phenol / chloroform mixture, then analyzed by Northern Blot. After migration on agarose gel, the RNAs are transferred to a nylon membrane, then hybridized with probes. marked with ³² P specific to the HSP70. The radioactive signals of the different bands are quantified by an imager.

In the case of stress, the results expressed are normalized with respect to the cadmium inducer.

Example 2: Effect of verbascoside on the secretion / maturation of collagen by visualization of a fluorescent antibody using the confocal microscopy technique.

The cells (normal human dermal fibroblasts) are cultured to confluence in a conventional DMEM 10% FCS medium, at 37 ° C., 5% CO 2.

Then they are treated with verbascoside (5.10-3%) for 2 times 72 hours.

After washing and saturation of the non-specific binding sites, the cells are treated with the polyclonal anti-collagen antibody I.

Observations are then made by confocal microscopy.

- Control cultures:

After labeling with an anti-collagen I antibody and revelation by epi-fluorescence, the extracellular matrix of the control cultures appears to be not very fibrous. The labeling of collagen is mainly located inside or associated with the surface of the cells (little deposit and poorly structured in fibers).

- Positive control:

TGF-beta is used as a positive control. Present naturally in the skin, one of its roles is to act on the expression of collagen genes and it leads to a sharp increase in the synthesis of pro- collagen. 16

After treatment with TGF-beta, the labeling of collagen is much more intense than in the controls.

The observation of the matrix deposited in the cells treated with verbascoside shows a modification compared to the control. More fibers are visible and they are larger and better organized.

Verbascoside has also shown an effect in cultures already treated with TGF-beta: the deposited matrix is denser than the case where cells are exposed to treatment with TGF-beta alone.

All these elements show us that the verbascoside is important for the secretion / maturation of collagen, for the organization of the extra-cellular matrix, therefore for the maintenance of the integrity and the quality of the dermis.

Example 3: Anti-wrinkle emulsion for the face (composition in percentage).

Verbascoside 0.1% (or dry extract containing 20%: 0.5%)

Demineralized water qs

Softener 0.05

Sequestering 0.025

Cocoate ethyl hexyl 5 Glycols 8

0.3 xanthan gum

Vegetable oils and waxes 4

Fatty alcohols 3.5

Silicone 5 Preservatives 1.9

Sun filters 2

Talc 2

Vitamins 0.4

Perfume 0.4 17

Example 4: Face and body emulsion (percentage composition).

Verbascoside: 0.1% (or dry extract containing 20%: 0.5%)

Demineralized water qs Propylene glycol 2 Moisturizer 2 Na 4 EDTA 0.025 Gelling agents 0.1 Preservative 0.2 Antioxidant 0.1 Vegetable oils 12 Silicone 3

Triglycerides 2 Softeners 0.1 Thickener 1.5 Emulsifiers 4 Ester 7 Perfume 0.3 Ethanol 3 Silica 1

Claims

1) Cosmetic use of a phenyl propanoid glycoside as an agent stimulating the synthesis of a heat shock protein by skin cells, said agent being incorporated in a cosmetic composition comprising one or more acceptable excipients. 2) Use according to claim 1, characterized in that the phenyl propanoid glycoside is the verbascoside. 3) Use according to one of claims 1 or 2, characterized in that the heat shock protein is HSP 70. 4) Use according to any one of claims 1 to 3, characterized in that the phenylpropanoid glycoside, more particularly verbascoside, is also an agent inhibiting collagenase and elastase and stimulating the maturation of collagen during its secretion by an action on hydroxylases. 5) Use according to any one of the preceding claims, characterized in that ^'said cosmetic or dermatological composition is intended to fight against the reduction of the integrity of the dermis due to the deterioration of the support fibers of the skin are collagen and elastin. 6) Ut ili sat ion according to which one of the preceding claims, characterized in that the phenyl propanoid glycoside, more particularly the verbascoside, is in the form of a pure molecule obtained by chemical synthesis, by enzymatic synthesis, or obtained by extraction and purification from fresh or dried plants. 7) Use according to one of the preceding claims, characterized in that the phenyl propanoid glycoside, more particularly the verbascoside, is in the form of a plant extract of which the concentration of phenylpropanoid glycoside, more particularly in verbascoside of between approximately 0 , 01 to 75%, preferably between 0.1 and 50%. 8) Use according to one of claims 6 or 7, characterized in that the phenyl propanoid glycoside, more particularly the verbascoside, is in the form of a plant extract chosen from the order of Tubiflorales. 9) Use according to one of claims 6 to 8, characterized in that the phenyl propanoid glycoside, more particularly the verbascoside, is in the form of a plant extract chosen from the genera Verbascum, Plantago, Verbena, Lippia, Fraxinus. 10) Use according to any one of the preceding claims, characterized in that the phenyl propanoid glycoside, more particularly the verbascoside, is free, encapsulated, vectorized, grafted. 11) Use according to any one of the preceding claims, characterized in that the phenyl propanoid glycoside, more particularly the verbascoside, is present in the cosmetic composition at a concentration of the order of 0.01 to 5%, preferably of 0.05 to 2%, by weight of the composition.