WO2010007247A2

WO2010007247A2 - Use of an aqueous or organic or aqueous-organic extract of microsorum

Info

Publication number: WO2010007247A2
Application number: PCT/FR2009/000854
Authority: WO
Inventors: Alain Meybeck; Raimana Ho; Taivini Teai; Phila Raharivelomanana
Original assignee: Am Phyto-Conseil
Priority date: 2008-07-16
Filing date: 2009-07-10
Publication date: 2010-01-21
Also published as: FR2933869A1; WO2010007247A3; FR2933869B1

Abstract

The invention relates to the use of an aqueous or organic or aqueous-organic extract of Microsorum for the manufacture of a cosmetic skincare or haircare composition, in particular for anti-ageing protection against the effects of oxidative stress or of ultraviolet radiation.

Description

Use of an aqueous or organic or hydro-organic extract of Microsorum.

The present invention aims to the use of an aqueous extract or ORGAM ^'that or hydro-organic Microsorum for the manufacture of a cosmetic composition for skin care or hair.

Metua pua 'a is the vernacular name of certain common ferns in Tahiti as in other Polynesian islands and in particular Microsorum scolopendria and Microsorum membranifolium of the Polypodiaceae family (Ho R et al, Communication at the IV International Symposium on Aromatic Plants and Médicinales d'Outre-Mer, Tahiti, French Polynesia, 2006, as well as Ho R et al, Communication at the XVIth International Ecdysone Workshop, Ghent, Belgium, 2006). These plants contain in particular ecdysteroids. For example Microsorum scolopendria contains ecdysone and 20-hydroxy-ecdysone alongside other ecdysteroids (Snogan E et al., Phytochem, Anal., 18, 441-450, 2007) and Microsorum membranifolium further contains 2- deoxy-20-hydroxyecdysone (Ho R et al., J Chromatogr Sci 46, 102-110, 2008). These ferns are used as ornamental plants. In addition, the fronds (leaves) and rhizomes of Metua pua 'a, are used as ingredients for the preparation of traditional remedies used in particular as a purgative or vermifuge (Pétard P, Useful plants of Polynesia, pp 78- 80. Raau Tahiti , 1986. Haere po no Tahiti).

Moreover, it is known that aging, and in particular the aging of connective tissues, such as the skin, is largely due to the appearance of "senescent" cells that secrete enzymes capable of degrading the extracellular matrix. pro-inflammatory cytokines, and growth factors (Campisi J and Adda di Fagagna, Nature Reviews Mol CeIl Biol 8, 729-740, 2007). It has indeed been established in cell culture replication studies, as well as in studies of skin cells of the elderly, that the skin cells can pass into a very specific state described as "senescent" by specialists. in Cell Biology. In this state, they can no longer reproduce, and they express a particular enzyme called "senally associated beta galactosidase" or SA beta, characterized by being active at pH 6 (Dimri GP et al, Proc Nat Acad Sci USA, 92: 9363-67, 1995). Use of an aqueous or organic or hydro-organic extract of Microsorum.

The object of the present invention is to use an aqueous or organic or hydro-organic extract of Microsorum for the manufacture of a cosmetic composition for skincare or hair care.

Moreover, it is known that aging, and in particular the aging of connective tissues, such as the skin, is largely due to the appearance of "senescent" cells that secrete enzymes capable of degrading the extracellular matrix. pro-inflammatory cytokines and growth factors (Campisi J and Adda di Fagagna F, Nature Reviews Mol CeIl Biol 8, 729-740, 2007). It has indeed been established in cell culture replication studies, as well as in studies of skin cells of the elderly, that the skin cells can pass into a very specific state described as "senescent" by specialists. in Cell Biology. In this state, they can no longer reproduce, and they express a particular enzyme called "senally associated beta galactosidase" or SA beta, characterized by being active at pH 6 (Dimri GP et al, Proc Nat Acad Sci USA, 92: 9363-67, 1995). Finally, it appears that the "senescent cells" in vivo may have adverse effects on the surrounding tissue (Funk WD et al, Exp CeIl Res.258: 270-8) and that it seems important to have means to avoid their appearance. .

It seems that it would be very interesting to have means to avoid the appearance of "senescent cells" that have lost the ability to reproduce, and have adverse effects on the surrounding tissues.

On the other hand, it is known that the skin of the exposed areas, and in particular the skin of the face, ages more rapidly because of the oxidative stress caused by ultraviolet radiation and atmospheric pollution which generate free radicals, peroxides, singlet oxygen and superoxide ion, which can degrade in the body proteins (enzymes, structural proteins ...), lipids of biological membranes, nucleic acids .... This is the reason why these skin areas should be protected by suitable cosmetic products.

Moreover, the enzyme heme-oxygenase or HO-I is known. This enzyme catalyzes the degradation of Theme to carbon monoxide, free iron, and biliverdin (Pae H-O et al, J. Clin Biochem Nutr 42, 197-203, 2008). Carbon monoxide CO is now recognized as an important mediator, particularly responsible for the photo-immunoprotection of Langerhans cells by UVA radiation in the skin (Allanson et al., J Invest Dermatol, 124, 664-50, 2005). Iron that could be dangerously pro-oxidant in the free state is immediately complexed by a specialized protein called ferritin. Biliverdin is rapidly converted by biliverdin reductase to bilirubin, a potent antioxidant (Stocker R et al., Science 235, 1043-6, 1987, Bach FH, Wien Klin Wochenschr, 114, 1-3, 2002). Thus, heme-oxygenase and ferritin play an important protective role in the body and it would be very advantageous to have substances that can activate their expression in cutaneous cells.

It is therefore an object of the present invention to stimulate, in cutaneous cells, the biosynthesis of certain proteins involved in their protection against oxidative stress, in particular HO-I heme-oxygenase and ferritin.

Another object of the invention is to provide a product whose active ingredient is natural. ^' These and other objects which will become apparent in the present invention are achieved by the use of an aqueous or organic or hydroorganic extract of Microsorum for the manufacture of a composition for the care of the skin. skin or hair likely to activate the expression in the cells of heme-oxygenase HO-I and ferritin.

Preferably, Microsorum is selected from the species commutatum, grossum, maximum, membranifolium, punctatum, rubidum, scolopendria, and more particularly membranifolium and scolopendria.

Advantageously, the composition for the care of the skin or hair is according to the present invention is a cosmetic product for protection against the effects of oxidative stress.

According to a preferred embodiment of the present invention, the Microsorum extract is a sling or rhizome extract made with one or more of the following solvents: water, methanol, ethanol, propanol, butanol, propylene glycol, butylene glycol.

Preferably, the final concentration expressed as Microsorum solids in the cosmetic composition is from 0.001 to 10% by weight and even more particularly from 0.05 to 1% by weight.

Advantageously, the cosmetic composition also contains at least one active substance such as an ascorbic acid derivative, a tocopherol, a tocopherol derivative, a retinol derivative, forskolin, caffeine, serine, glycerol, resveratrol, Centella extract, Ginseng extract, Notoginseng extract, Rhodiola extract, Eleutherococcus extract, Glycyrrhiza extract, Scutellaria extract, Griffonia extract, arbutin, kojic acid, a UV filter. Preferably, the cosmetic composition is in the form of an oil-in-water emulsion, a water-in-oil emulsion, a multiple emulsion, a lotion, a gel, a suspension of liposomes, a suspension of lipid spherules.

Advantageously, an aqueous or hydro-ethanolic Microsorum extract is used for a composition for the care of the skin or hair as a composition for the oral route.

Thus, one object of the present invention is the production of new cosmetic products for protecting the skin against aging, characterized in that that they contain Microsorum extracts such as Microsorum scolopendria, or Microsorum mβmbranifolium, and more particularly extracts of rhizome and slingshot (leaf): it has in fact been surprisingly found that a particular fraction of an extract of Microsorum is likely to protect the skin cells against the appearance of "senescence".

In order to obtain the extracts, a solvent is selected which can extract the relatively polar compounds without extracting too many free sugars or very hydrophobic substances. Methanol is a good compromise, but you can also use a water-ethanol mixture or hot water. In the case of a hot methanol extract, it is possible after evaporation to carry out an extraction with an apolar solvent to remove colored products such as carotenoids and degradation products of chlorophyll still present in the leaves. In the case of extraction with hot water, it is possible after drying to partition with butanol in order to collect the less polar compounds and to get rid of the sugars. For the production of cosmetic compositions, dispersions of a fatty phase are generally chosen in water or vice versa, so as to gather in the formula a maximum of ingredients intended to give the product easy spreading properties. , a given consistency, a pleasant final touch, a good stability, a protection against microbial contaminations ... In certain cases we will prefer more complicated formulations such as multiple emulsions or liposomes, or on the contrary simpler such as lotions or aqueous gels.

The following description, which is in no way limiting, relates in particular to exemplary embodiments which will enable those skilled in the art to better understand the present invention.

Example 1: Obtaining a Gross Extract from Microsorum scolopendria.

50 g of dried fronds (leaves) from Microsorum scolopendria {M. scolopendria) are extracted with soxhlet per 100 ml of methanol for two times 7 hours.

The methanol extract is evaporated on a rotary evaporator to give 10.25 g of dry crude extract. Example 2: Obtaining a Butanolic Fraction of the Gross Microsorum scolopendria Extract (M. scolopendrid). 146.5 g of dry methanol crude extract as in Example 1 are prepared from 715 g of dry Microsorum scolopendria fronds.

This dry extract is taken up in one equivalent of methanol, one equivalent of chloroform, and one equivalent of water. This gives two phases that are allowed to settle. The polar phase is separated and concentrated to give 28.21 g.

This dry polar phase is taken up in 200 ml of water before the addition of 200 ml of n-butanol.

The organic phase is separated and evaporated to give 1.25 g. This dry fraction is then taken up in 2 ml of a 3: 1 water-ethanol mixture and passed for purification on a polyamide gel column (47 × 3 cm) with the same hydro-alcoholic mixture as solvent. The first 300 ml solvent volume corresponds to the dead volume of the column and is not retained. The second volume of 300 ml of eluate is collected and evaporated to give 500 mg of dry Butanolic Fraction of Microsorum scolopendria extract.

Example 3 Obtaining an Ethanolic Fraction of Microsorum scolopendria

50 g of fronds (leaves) of Microsorum scolopendria are macerated for 24 h in 100 ml of a mixture of water and ethanol 1 to 1 by volume. After filtration, the extract is evaporated to dryness. The dry extract is taken up in 50 ml of ethanol at 96 ° to prepare an ethanolic fraction. It is filtered through celite, and the ethanol is evaporated to obtain the dry ethanolic fraction of Microsorum scolopendria.

EXAMPLE 4 Obtaining a Microsorum membranifolium Raw Extract 30 g of dried fronds (leaves) of Microsorum membranifolium are extracted cold for one day with 100 ml of methanol. The extract is then dried by evaporation of the solvent in a rotary flask to give 6 g of crude extract of M. membranifolium.

Example 5 Obtaining a crude extract of Microsorum membranifolium.

100 g of dry rhizomes of Microsorum membranifolium are crushed, then extracted with 300 ml of a mixture of water and ethanol 1/1 by volume. After filtration, the extract is evaporated to dryness.

Example 6: Qualitative test of activity on cutaneous cells of Microsorum scolopendria extracts. Normal human fibroblasts are cultured for 48 h in medium

DMEM supplemented with 10% fetal calf serum. Then aliquots of culture were added via a stock solution in DMSO, 10 mg / ml of M. scolopendria crude extract of Example I ₅ or 2 ug / ml of the butanol fraction from M. scolopendria of Example 2, and incubated for 24 h, as well as control cultures without extract.

After 24 hours of contact with the extract or the fraction to be tested, a differential analysis of the expression of the genes in the fibroblasts is carried out by means of alignments of complementary DNA sequences ("c-DNA arrays") deposited on a polyamide membrane. For this, total RNA messengers extracted and then the "reverse transcribed" by using a mixture of primers corresponding to the c-DNA deposited on the membrane in the presence of labeled deoxynucleotide triphosphate to radioactive phosphorus ³³ P. Thus labeled DNA sequences are prepared, and can then be "hybridized" to the target c-DNAs deposited in excess on the membrane. After washing, the c-DNA alignments are revealed by autoradiography and the resulting spots are subjected to an image analysis which by comparison with a series of control genes, gives a semi-quantitative assessment of genes that are over-expressed or under-expressed. -expressed in the cells.

It was thus found (Table 1) that the heme-oxygenase HO-I and ferritin genes were overexpressed in the fibroblast cultures incubated in the presence of the M. scolopendria extract, but not in such a manner. significant in the culture treated with the butanolic fraction. Table 1

EXAMPLE 7 Quantitative Activity Test of M. scolopendria Extracts on Cutaneous Cells

To confirm the results of Example 5, quantitative RT-PCR analysis was performed. For this, the total messenger RNAs of the culture treated with the crude M. scolopendria extract of Example 5 were subjected to a polymerase chain reaction (PCR) in a Roche Molecular Systems Light Cycler®. Ihc. This system includes a thermo-cycler optimized for extremely fast PCR reactions, and a fluorometer to evaluate at 521 nm, the SYBR Green I dye which is specifically inserted between the DNA double helices at each cycle of duplication. The following "primer" couples were used:

Heme-oxygenase I (HO-I, gene code bank NM-002133)

Ferritin (FTH1, gene code bank NM-002032).

The glyceraldehyde-3-phosphate dehydrogenase (G3PDH) gene was used as a reference marker. The results expressed in% of the expression of the gene considered in the culture treated with the raw extract of M. scolopendria, compared to the non-treated control culture are given in Table 2.

Table 2

Thus the crude extract of Microsorum scolopendria activates the expression of the genes of heme-oxygenase and ferritin in skin fibroblasts.

EXAMPLE 8 Test for Prevention of Cutaneous Cell Senescence by the Butanolic Fraction of an Extract of Microsorum scolopendria (F Bu from M.S.)

This test demonstrates the attenuation by the butanolic fraction of Example 2 of the damage caused to fibroblasts in culture by repeated oxidative stress resulting from repeated exposure to ultraviolet radiation.

Dermal fibroblasts of the same BJ strain as used by Chainaux F. et al., J. Biochem, Cell Biol 34 (11): 1331-9,2002) are cultured by conventional methods.

The absence of cytotoxicity of the butanol fraction of Example 2 over a period of 24 hours is verified in the range 50 to 250 μg / ml.

For 5 days, the fibroblasts are exposed once a day to a dose of 200 B / cm ² of UV-B.

24 hours before the first UV treatment, and following each irradiation, the culture medium is replaced with medium containing 50, 125, and 250 μg / ml of fraction F Bu M.s ..

Before each new irradiation, these media are replaced by a medium containing no fraction F Bu M.s ..

After the last irradiation, the cells are left for 72 hours in contact with medium supplemented with F Bu of M.s, renewed every 24 hours.

At the end of this last period of contact, the beta-galactosidase activity at pH6 (beta-galactosidase associated with senescence or SA beta-gal) is evaluated according to the method of Dirnri GP et al (already cited), on at least 400 cells for each concentration in F Bu of Ms.

The experiment was performed twice.

The average values of the percentages of cells expressing beta-gal SA are summarized in the Table below. Table 3

It can be seen that: - the damage caused by repeated exposure to ultraviolet B rays has led to a significant increase in fibroblasts exhibiting the characteristic beta-galatic activity of cells that can no longer reproduce (termed "senescent").

the treatment of the cells with the butanolic fraction of Microsorum scolopendria of Example 2 before and after the irradiations, made it possible to attenuate the damage caused by the ultraviolet rays as early as the concentration of 50 μg / ml, which results in a number of "senescent" fibroblasts which remains lower than that of cultures that have not undergone any irradiation.

It thus appears that the use of the extract fraction of Microsorum scolopendria makes it possible, according to the invention, to attenuate the damage caused to cutaneous cells by repeated exposure to oxidative stress.

Composition No. 1: anti-aging skin care cream.

The procedure is as for the production of a conventional oil-in-water emulsion, that is to say that a hydrophobic phase and an aqueous phase containing each of the raw materials intended to impart the product to the product are mixed with stirring at 85 ^° C. Usual properties: stability, safety, easy spreading, nice final touch, etc. The mixture is then cooled with stirring and at 50 ^° C., 0.5% of crude Microsorum scolopendria extract is added as in Example 1. The cooling is continued with stirring until the ordinary temperature is reached.

This cream is applied to the face, neck and hands, preferably in the morning so that the principle stimulating the natural defenses has time to penetrate to the living cells, to be in contact in the middle of the day when the intensities solar irradiation are at their maximum.

Composition No. 2: Cosmetic cream for protection against oxidative stress. As above, an oil-in-water emulsion containing retinol palmitate is produced; during cooling to 45 ° C., fractional extract of Microsorum scolopendria as in Example 2 is added, together with a glycolic extract of Scutellaria baicalensis in an amount sufficient to arrive at the concentrations by weight in the final composition of : - fractional extract of Microsorum scolopendria 0.2%

retinol palmitate 0.05%

- glycolic extract of Scutellaria baicalensis 2%. This formulation is a day cream.

Composition N ° 3: Cosmetic cream for anti-aging protection and filter protection.

As in Composition No. 1, an oil-in-water emulsion with an addition of titanium oxide sufficient to obtain an SPF protection factor equal to 6 is carried out, then an extract of Microsorum scolopendria is added as in Example 1. 1 with stirring at 45 ° C in an amount sufficient to obtain 0.4% by weight extract in the final preparation. .

The product is intended to be applied in the morning on uncovered skin areas, and if possible, the application should be renewed mid-day. Composition N ° 4: Anti-aging cosmetic gel for sensitive skin.

A hydroalcoholic acrylic gel is titrated with 0.3% by weight of Microsorum membranifolium extract as in Example 4 and containing in addition 2% by weight of glycolic acid extract of Glycyrrhiza glabra.

Composition N ° 5: Moisturizing cream for anti-aging protection.

As in Composition 1, an oil-in-water emulsion containing 0.5% of Microsorum membranifolium extract of Example 5, 3% of glycerol and 1% of serine is prepared, these three constituents being added to the manufacture during cooling, when the temperature has reached 50 °.

Composition N ° 6: Anti-hair loss lotion

A hydroalcoholic hair fall protection lotion containing 0.5% of Microsorum scolopendria extract as in Example 1 and 0.1% of Panax Notoginseng rhizome saponins is prepared.

Composition No. 7: Dietary supplement to protect the skin before exposure to the sun or pollution. 5 mg tablets per tablet of dry ethanolic fraction of a 1: 1 ethanolic extract of Microsorum scolopendria are prepared by conventional techniques as in Example 3.

These tablets should be taken with a little water during the morning and mid-day meals, one to two tablets per dose depending on the intensity and duration of exposure expected.

Composition N ° 8: Food supplement drink to protect the skin during sun exposure.

A coconut water drink containing 0.01% by weight of dry ethanolic fraction of Microsorum scolopendria extract is prepared as in Example 3.

This drink is presented in doses of 100 ml. It is advisable to drink a dose each morning at breakfast.

Claims

1. Use of an aqueous or organic or hydro-organic extract of Microsorum for the manufacture of a composition for the care of the skin or hair capable of activating the expression in the cells of heme-oxygenase HO- I and ferritin.

2. Use according to claim 1, such that Microsorum is chosen from the species commutatum, grossum, maximum, membranifolium, punctatum, rubidum, scolopendria, and more particularly membranifolium and scolopendria.

3. Use according to one of claims 1 or 2 such that the composition for the care of the skin or hair is a cosmetic product for protection against the effects of oxidative stress.

4. Use according to one of claims 1 to 3 such that the Microsorum extract is a sling or rhizome extract made with one or more of the following solvents: water, methanol, ethanol, propanol, butanol, propylene glycol, butylene glycol.

5. Use according to one of claims 1 to 4 such that the final concentration expressed as Microsorum solids in the cosmetic composition is from 0.001 to 10% by weight.

6. Use according to claim 5 such that the final concentration expressed as microsorum solids in the cosmetic composition is more particularly from 0.05 to 1% by weight.

7. Use according to one of claims 1 to 6 such that the cosmetic composition also contains at least one active substance such as a ascorbic acid derivative, a tocopherol, a tocopherol derivative, a retinol derivative, forskolin, caffeine, serine, glycerol, resveratrol, a Centella extract, a Ginseng extract, a Notoginseng extract, a Rhodiola extract, an extract of Eleutherococcus, an extract of Glycyrrhiza, an extract of Scutellaria, a Griffonia extract, arbutin, kojic acid, a UV filter.

8. Use according to one of claims 1 to 7 such that the cosmetic composition is in the form of an oil-in-water emulsion, a water-in-oil emulsion, a multiple emulsion, a lotion, a gel, a suspension of liposomes, a suspension of lipid spherules.

9. Use of an aqueous or aqueous ethanol extract Microsorum according to claims 1 or 2 such that the composition for the care of the skin or hair is a composition for the oral route.