EP2437721A2

EP2437721A2 - Pine pco ester cosmetic composition

Info

Publication number: EP2437721A2
Application number: EP10734197A
Authority: EP
Inventors: Nicolas Huguet
Original assignee: Action Pin SAS
Current assignee: IXXI
Priority date: 2009-06-02
Filing date: 2010-06-01
Publication date: 2012-04-11
Also published as: JP2012528838A; WO2010139887A2; CA2763968A1; US20120142767A1; WO2010139887A3; EA025244B1; FR2945940A1; EA201101682A1; FR2945940B1

Abstract

The invention relates to a cosmetic composition for repairing and optionally preventing the effects of skin ageing, containing an extract of Pinus pinaster bark in which the procyanidolic oligomers (PCOs) are esterified with a fatty acid. Said compositions are particularly useful for increasing collagen synthesis, for soothing the skin, or further for protecting the skin against free radicals.

Description

COSMETIC COMPOSITION BASED ON PIN OPC ESTER

The invention relates to a cosmetic composition for repairing and possibly preventing the effects of aging of the skin based on a maritime pine bark extract whose procyanidolic oligomers (OPC) are esterified with a fatty acid. These compositions are useful in particular to increase collagen synthesis, to soothe the skin, or to protect the skin from free radicals.

ProCyanidolic Oligomers (PCOs), also called proanthocyanidins or proanthocyanidic oligomers, are natural molecules that are widely used in nature. Indeed, they are found in vegetables, fruits, cereals but also in grape seeds and pine bark. The nutritional and physiological interest of these molecules is related to their antioxidant properties and their benefit on the cardiovascular system.

OPCs belong to the chemical family of polyphenols and more particularly to the class of flavonoids, compounds known for their antioxidant properties. The OPCs consist of one or more repeating monomeric units of flavan-3-ol, which units are independently selected from catechin, or epicatechin or epicatechin gallate. These units are usually repeated 2 to 4 times, the most common OPCs being generally in dimer form.

Biochemical structure of a UCI

(catechin trimer)

However, the phenolic functions of OPCs make these compounds particularly sensitive to various degradation factors such as light, air and acidic or basic pH. This instability is, in the majority of cases, a brake on the exploitation of their beneficial effects. To stabilize these compounds, it has notably been proposed to esterify the polyphenol oligomeric extracts with long chain hydrocarbon fatty acid derivatives (FR 2 723 943). The inventors have now demonstrated that a pine bark extract whose procyanidolic oligomers (OPC) are esterified with a fatty acid, had particularly interesting cosmetic properties. It has thus been demonstrated that OPC esters extracted from pine bark make it possible to increase collagen synthesis by human dermal fibroblasts, and thus to repair the effects of skin aging. It has also been demonstrated that these OPC esters also have anti-inflammatory and anti-radical properties, making them particularly useful for soothing the skin and preventing the effects of skin aging.

The present invention thus relates to a cosmetic composition for repairing the effects of skin aging comprising: a) procyanidolic oligomers (PCOs) extracted from pine bark, said oligomers being esterified with at least one fatty acid; and b) a cosmetically acceptable vehicle.

According to a preferred embodiment, the cosmetic compositions according to the invention comprise: a) a pine bark extract whose procyanidolic oligomers (OPC) are esterified with at least one fatty acid, and b) a cosmetically acceptable vehicle.

By "cosmetically acceptable vehicle" is meant a vehicle adapted for use in contact with human and animal cells, in particular the cells of the epidermis, without toxicity, irritation, undue allergic response and the like, and proportionate to an advantage ratio. / reasonable risk.

Extracts of pine bark, especially maritime, contain a large variety of procyanidolic oligomers, including catechin monomers, and taxifoliol, as well as procyanidol oligomers of 2 to 5 flavan-3-ol units, the monomers and the dimers being predominantly present.

The composition of pine bark extracts is qualitatively and / or quantitatively different from that of other extracts of plant origin, in particular grape seed extracts, which are found in particular in the trade under the name VITAFLAVAN ®. More specifically, the procyanolidic oligomers extracted from pine bark comprise in particular taxifoliol, taxifoliol glucoside, ferulate glucoside and various phenolic acids, which are not present in grape seed extracts. Unlike these, they do not include epicatechin or epicatechin gallate. OPCs generally account for about 70% by weight of the total weight of pine bark extract. Maritime pine bark extracts are commercially available

(OLIGOPIN®, DRT). They can also be obtained by a conventional extraction process such as that described in patent FR 998 508.

The pine bark extract may be in particular a French maritime pine (Pinus pinaste®) extract, such as the Landes pine, According to a preferred embodiment of the invention, the procyanidolic oligomers (OPC) contained in the extracts of maritime pine bark are esterified with a fatty acid according to the process described in the patent application FR 2 723 943. More particularly, the OPC are esterified according to the process comprising the steps of: a) contacting the extract of maritime pine bark in a non-solvent liquid medium for said extract but solvent for the esters to be obtained, so as to obtain a suspension; b) adding to said suspension at least one low-boiling aliphatic tertiary amine, in the presence of a catalytic amount of at least one organic base other than pyridine, and c) introducing into this mixture from least one fatty acid chloride, the reaction mixture being stirred at a temperature below 4O ⁰ C and then concentrated by evaporation to obtain an extract including the OPC are esterified.

Preferably, the OPCs are esterified with saturated fatty acids, in particular palmitic acid and stearic acid, more preferentially palmitic acid.

The application of a cosmetic composition according to the invention is carried out topically.

The composition is intended more particularly for the treatment of the skin and may be in the form of ointment, cream, oil, milk, ointment, powder, impregnated buffer, solution, gel, spray, lotion, suspension, soap.

The compositions according to the invention can be cosmetic compositions in the form of oil-in-water or water-in-oil emulsion, or multiple emulsions, microemulsion, hydroalcoholic gel, cream, oil of hydroalcoholic lotion. Preferably, the cosmetic compositions according to the invention may comprise from 0.1 to 2.5% of said esterified extract expressed by weight relative to the total weight of the composition, preferably from 0.2 to 1.0%.

The cosmetic compositions according to the invention are particularly useful for increasing collagen synthesis, in particular by human dermal fibroblasts. They are also advantageously useful for soothing the skin, and / or for protecting the skin against free radicals. They are thus particularly useful for preventing the effects of skin aging, especially photoaging.

The invention also relates to the use of a cosmetic composition comprising: a) procyanidolic oligomers (PCOs) extracted from pine bark, said oligomers being esterified with at least one fatty acid; and b) a cosmetically acceptable vehicle for repairing the effects of skin aging. The present invention also relates to a cosmetic treatment method, comprising the application, especially topical, of a cosmetic composition as defined above.

Thus, the present invention relates to a cosmetic treatment method, for repairing the effects of skin aging, comprising the application of a cosmetic composition comprising procyanidolic oligomers (PCOs) extracted from pine bark, said oligomers being esterified with at least a fatty acid; and a cosmetically acceptable vehicle.

The present invention also relates to a cosmetic treatment method, for repairing the effects of skin aging, comprising the application of a cosmetic composition comprising a pine bark extract whose procyanidolic oligomers (OPC) are esterified with at least one acid fat, and a cosmetically acceptable vehicle.

The following examples illustrate the invention without limiting it. EXAMPLES

Example 1 Preparation of an extract of maritime pine bark containing esterified OPCs

The pine bark extract (Oligopin ^®) is esterified with palmitic acid according the method described in patent application FR 2,723,943.

In the examples which follow, the terms "pine OPC esters" refer to the esterified pine bark extract according to the present example 1.

Example 2

Study of the cytotoxic activity of pine OPC esters on a cell monolayer in culture

1. Objective of the study

The purpose of this study is to evaluate, by an in vitro test, the ability of the pine OPC esters prepared according to Example 1 to induce cytotoxic effects on a monolayer of human fibroblasts.

2. Principle of the study

The objective of the study is to evaluate the ability of pine OPC esters to induce cytotoxic effects on a primary culture of normal human fibroblasts. After application of the product to cells for 24 hours, cell viability is assessed by measuring the activity of mitochondrial succinate dehydrogenase in living cells. This enzyme transforms MTT (3 (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) into blue formazan crystals. After dissolution of these crystals, a spectrophotometric reading is carried out. The measured absorbances are proportional to the number of living cells.

3. Test system

The cells used are fibroblasts of human skin tissue (prepuce Caucasian child), passing 1. 4. Conduct of the study

Reference elements:

The following controls are included at each analysis

- Negative control: complete culture medium

- Positive witness: SDS

Definition of series:

Six dilutions of each sample, prepared in 0.005% tween 60 and 0.05% cottonseed culture medium, are tested at 5 test wells per concentration. The reference elements are tested on 5 wells.

5. Expression and interpretation of the results

Absorbance is measured at 540 nm. The results are expressed as a percentage of mortality compared to the negative control:

. ... Abs negative control - Abs sample. _nn

Vc mortality = x100

Abs negative control

6. Results

The positive control has a mortality greater than 30%, this validates the test.

7. Conclusion

Under the experimental conditions adopted, the pine OPC esters according to Example 1 do not exhibit a significant cytotoxic effect. For all efficacy studies, the maximum concentration used should be 1000 μg / ml.

Example 3 Screeninq Study of the Anti-inflammatory and Anti-Radical Potential

1. Principle of the study

A human epidermis is reconstituted in vitro in culture inserts. A disk of filter paper is deposited on the "stratum corneum" on which the substance under test has been deposited (preventive treatment). 24 hours later, the UVB is irradiated and then the substance is again applied to the test (curative treatment). 24 hours later, we realize:

- a cytotoxicity study (MTT),

a pro-inflammatory mediator of NL-I, in the culture medium, an MDA (malonaldehyde) assay (end product formed during the process of lipid peroxidation induced by the formation of radical species, or of species activated by oxygen).

2. Identification of the test substance OPC esterified according to Example 1 in solution at 1% (w / w) in the solvent E (50

% isopropanol - 50% isopropyl myristate in w / w)

3. Protocol

3.1. Preparation of the reagent system: reconstructed human epidermal sheets:

- Culture of human keratinocytes in the ^3rd pass free of mycoplasma

The keratinocytes are inoculated at the density of 100 × 10 ³ cells / cm ² in inserts whose bottom consists of a polycarbonate membrane (porosity 0.4 μm-1 cm ² ). The cells are incubated at 37 ^° C. in a humid atmosphere containing 5% (v / v) of CO ₂ for 24 hours, in the "immersed" situation in the culture medium: MCDB / 153 supplemented with EGF (5 ng / ml) mL), insulin (5 μg / mL), hydrocortisone (0.5 μg / mL), BPE (70 μg / mL). - The culture medium is replaced by the same culture medium supplemented with 1 mM Ca ⁺⁺ to induce the stratification of the monolayer in "immersed situation" for 6 days. The culture medium is changed every three days.

- The epidermal sheet is set "position emerged" the ^6th day, that is to say that it is not nurtured only through the basal cells, the stratum corneum being exposed to air. The culture medium is changed every three days.

3.2. Preparation of the substance under test:

At the 14 ^th day of incubation, the culture medium is changed and replaced by the same culture medium supplemented with 1% antibiotics (10,000 U Penicillin - 10,000 μg / mL Streptomycin - 25 μg / mL Amphotericin) 20 μL of the test substance (3 non-cytotoxic concentrations), or 20 μL of the negative controls (culture medium) or solvent controls are applied under sterile conditions on a filter paper disc (Whatman # 3MMChr) covering the surface of the reconstructed epidermis, the side on which the test substance was deposited, and the controls in contact with the stratum corneum.

3.3. Definition of series:

The following series are implemented in triplicate:

- 3 test substance series (5, 50 and 500 μg / mL) consisting of 3 epidermis each (to be irradiated),

- 1 absolute negative control series (not irradiated),

- 1 absolute negative control series (to be irradiated),

1 solvent control series (non-irradiated),

1 solvent control series (to be irradiated), 1 positive control series 1 (Vit E: 200 μg / mL) (to be irradiated),

1 positive control series 2 (Indomethacin: 10 μg / ml) (to be irradiated),

The first eight series are incubated for 24 hours at 37 ^° C. in a humid atmosphere containing 5% (v / v) of CO ₂ (preventive treatment for the treated series). The ninth series, corresponding to the positive control 2 (Indomethacin), consists of depositing 20 μl of a solution of indomethacin at 10 μg / ml on a disk of filter paper (Whatmann No. 3 MMChr) deposited in contact with the epidermis reconstructed and incubated for 3 hours at 37 ° C, 21 h after the implementation of the previous series.

Then, the filter paper discs are carefully removed and all series are irradiated with UVB (0.6 J / cm ² ) (Spectroline model X-15N / F, λ = 312 nm) except the series corresponding to the absolute negative control and the solvent witness.

The curative treatment is then carried out as described above for the preventive treatment over a period of 24 hours, except for the positive control series 2

(Indomethacin).

3.4. Estimation of the cell population (MTT ^* test): The MTT test is performed according to the conditions described in the Hausen M. B, Nielsen SE and Berg K. Re-examination and further development of a precise and rapid dyemethod for measuring cell growth / cell kill. J. Immunol. Methods. 1989, 19, 203; and Mosmann T., Rapid Colorimetric Assay for Cell Growth and Survival: Application to Proliferation and Cytotoxic Assays, J. Immunol. Methods. 1983, 65, 55-63. At the end of the incubation time chosen, the Whatman paper disc is carefully removed, the "stratum corneum" rinsed, and the porous membrane bearing the epidermal sheet is detached from the insert using a scalpel. The culture media used for the determination of IL-1 α I ¹ and MDA.

The epidermal leaflet is: - rinsed with PBS (2 times),

- incubated for 3 hours at 37 ⁰ C, in the dark, in the presence of 2 mL of MTT (3- {4,5-dimethylthiazol-2-yl} - 2,5 - diphenyl tetrazolium) at 0, 5 mg / ml of culture medium,

rinsed with PBS (2 times). Purple formozan crystals produced by the cleavage of MTT by mitochondrial enzymes are dissolved in 2 mL of DMSO with gentle shaking for one hour at room temperature.

For each sheet, 200 μL of the solution obtained is pipetted into a 96-well plate to perform spectrometry with a plate reader (FL600 - BIO-TEK) at λ = 540 nm. 3.5. Assay of Tl L-1 α:

The dosage of I L- 1 α is performed in the collected culture media (n = 3) diluted 1/10 ^th and 1 / 20th in accordance with the protocol described in the assay kit.

3.6. Dosage of MDA:

Preparation of the standard range of MDA:

10 μM stock solution of malonaldehyde bi [dimethylacetal] in 1% (v / v) sulfuric acid (2 hours at room temperature),

dilutions are carried out between 0.1 and 10 μM (0.1 - 1 - 2.5 - 5 and 10 μM). 500 μL of culture medium (or 500 μL of standard range) are vigorously mixed with the Vortex to 1 mL of a "TCA, TBA, HCI" (TCA (trichloroacetic acid) 0.15% (w / v) solution, of 0.35% (w / v) TBA (2-thiobarbituric acid), 0.25 M HCl (heated at 100 ° C for 15 minutes to dissolve the TBA, and then allowed to cool).

The mixture is incubated for 15 minutes at 100 ° C. and the OD measured at λ = 535 nm against a blank consisting of the "TCA, TBA, HCI" solution.

4. Results

1 '

Table 1

^* UA: arbitrary units

Table 2

UA arbitrary units

4.1. cytotoxicity

• Irradiated absolute negative control: A decrease of 37% (P <0.05) of the OD (UA MTT) compared with the absolute negative control is observed, which corresponds to the cytotoxic effect generally obtained for the dose of irradiation used (0.6 J / cm ² ) and validates the irradiation.

• The irradiated absolute negative control, treated with indomethacin 3 hours before irradiation: there is no protective effect of indomethacin with regard to cytotoxicity.

• The irradiated negative control, treated with Vitamin E 24 hours before irradiation: a protective effect of Vitamin E is observed since the cytotoxic effect is not more than 13% (NS), about one-third of that observed in the absence of Vitamin E.

• The solvent control: it did not significantly modify cell proliferation. • The irradiated solvent control: it is not significantly different from the irradiated absolute negative control.

• The substance under test: a protective effect is observed. For 500 μg / mL, the cytotoxic effect is 17% (NS).

4.2. Study of the assay of I ¹ IL-1 α (Table 1):

• Irradiated absolute negative control: The level of IL-1α in the culture medium is very important: + 290% (P <0.02) compared to the absolute negative control. This result validates the reactive system which appears particularly reactive.

Irradiated absolute negative control treated with indomethacin 3 hours before irradiation: indomethacin used as a preventive inhibits partially (-20%) the synthesis and secretion of IL-1α in the culture medium. Although not statistically significant, this effect corresponds to the values usually obtained.

• Solvent control: it is not significantly different from the absolute negative control. • Irradiated solvent control: as for the irradiated absolute negative control, the level of IL-1α is greatly increased. This increase is not significantly different from that observed for the irradiated absolute negative control.

• The substance under test (pine OPC esters): a decrease in the level of IL-1 α in the culture medium is observed; this effect is particularly clear for the highest concentration (500 μg / mL): - 41% (P <0.02) relative to the irradiated solvent, which is twice the indomethacin-induced inhibition.

4.3. MDA study (Table 2):

• The irradiated absolute negative control: the MDA level is increased by 175% (P <0.02) compared to the absolute non-irradiated negative control, which validates the reagent system.

• The absolute negative control irradiated, treated with Vitamin E before and after irradiation: the increase of the MDA rate is only 58% (P <0.05), which represents one third of the rate observed for the irradiated absolute negative control untreated. • The non-irradiated solvent control: it has no significant effect on the rate of

MDA, compared to the absolute negative control. • The irradiated solvent control: it has no significant effect on the level of MDA, relative to the absolute negative control irradiated.

• The substance under test (pine OPC esters): a decrease in the level of MDA in the culture medium is observed for all the concentrations studied. For 50 μg / mL, a 43% inhibition is observed with respect to the UVB irradiated solvent control.

5. Conclusion

For the non-cytotoxic concentrations studied, 5, 50 and 500 μg / mL, the substance under test, OPC esterified according to Example 1, in solution at 1% (w / w) in the solvent E (50% isopropanol - 50% isopropyl myristate in w / w):

- protects the irradiated epidermis from UVB-induced cytotoxicity;

decreases the level of IL-1 α secreted in the culture medium after irradiation with UVB: 42% relative to the irradiated solvent control for 500 μg / ml; decreases the level of MDA secreted in the culture medium: 35% to 45%.

These "screening" results show anti-inflammatory and even antiradical activity.

Positive and negative controls validated the experiment.

Example 4

Study of the effect of a test element (pine OPC esters of Example 1) on the synthesis of collagen by human fibroblasts

1. Objective of the study The purpose of this study is to evaluate, by an in vitro test, the effect of the pine OPC esters according to Example 1 on the synthesis of collagen by a culture of human primary fibroblasts. .

2. Principle of the study

Fibroblasts are the main cells of the dermis. They are specialized in the synthesis of two types of protein fibers: collagen fibers and elastin fibers constituents of the extracellular matrix. Collagen is 70% of the dermis proteins and gives it resistance to tension and traction. The objective of this study is to evaluate the effect of the test element on collagen synthesis after a 24-hour contact with a normal human fibroblast monolayer. 3. Test system Cells used:

The cells used are fibroblasts of human skin tissue child (boy, Caucasian), passing 2. The fibroblasts are prepared and updated according to the operating procedure in force.

Reference elements:

The following controls are included at each analysis: - Negative control: complete medium - Positive control: TGFβ 10ng / ml

Definition of series:

Three non-cytotoxic dilutions of each sample, prepared in culture medium at 0.005% Tween 60 and 0.05% Cottonseed Oil, were tested at 3 wells per concentration.

The reference elements are also tested in triplicate.

4. Conduct of the study

Contacting the cells with the product under study: The cells are inoculated at a rate of 50,000 cells / cm ² . After 24 hours of culture (37 ^<€, 5% CO ² J, they were incubated with the product under consideration and the reference element for 24 hours in an environment of 1% FCS Determination of collagen.:

The collagen is assayed in the extracellular matrix and in the culture medium using the Sircol assay kit, Biocolor Ltd, Ireland, according to the protocol described in the kit.

The assay is performed using the Sirius red dye (Direct Red 80) which has a specific affinity for the triple helix structure (GIy-XY) _n of native collagen. The absorbance of the dye-collagen complex is read in the spectrophotometer at

540 nm.

A standard curve is carried out between 0 and 50 μg / ml of type I collagen.

Measurement of cell density: The cell density is evaluated under the same conditions as above, by measuring the activity of succinate dehydrogenase mitochondrial living cells. This enzyme transforms MTT (3 (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) into blue formazan crystals. After dissolution of these crystals, a spectrophotometric reading is carried out. The measured absorbances are proportional to the number of living cells.

5. Expression and interpretation of the results

For the evaluation of the cell density, the measured absorbance is expressed in units of MTT (U MTT). It is proportional to the amount of cells present in each well.

The concentration of collagen is expressed in μg of collagen / ml / U MTT in the extracellular matrix and in the culture medium.

Results

Vehicle: culture medium at 0.005% tween 60 and 0.05% cottonseed oil

TGF β1 at 10 μg / ml significantly stimulates (38%) the total synthesis of collagen. This result validates the model used. Stimulation is particularly effective at the level of the cell matrix (88%).

Regarding the effect of the product under test, we can note a very significant stimulation of total collagen synthesis with a maximum of more than 175% for a concentration of 50 μg / ml of the product.

It can also be seen that the product has an effect essentially on the secretion of collagen contrary to what can be observed for the positive control (TGF β1).

The effect is maximum for the concentrations 200 and 50 μg / ml.

At 500 μg / ml, the effect remains very marked but it is less important. At 5 μg / ml, the product has no effect. This type of response, in "bell", is very often observed in this type of study.

6. Conclusion

In the experimental conditions used, the pine OPC esters according to Example 1 have a significant effect on the synthesis of collagen.

Claims

1. Cosmetic composition for repairing the effects of skin aging comprising: a) procyanidolic oligomers (PCOs) extracted from pine bark, said oligomers being esterified with at least one fatty acid; and b) a cosmetically acceptable vehicle.

2. Cosmetic composition according to claim 1, for further preventing the effects of skin aging.

3. Cosmetic composition according to claim 1 or 2, comprising: a) a pine bark extract whose procyanidolic oligomers (OPC) are esterified with at least one fatty acid; and b) a cosmetically acceptable vehicle.

4. Cosmetic composition according to any preceding claim, wherein the fatty acid is palmitic acid.

5. Cosmetic composition according to any one of the preceding claims, intended for topical use.

6. Cosmetic composition according to claim 5, in the form of ointment, cream, oil, milk, ointment, powder, impregnated buffer, solution, gel, spray, lotion, of suspension, soap, or shampoo.

7. Cosmetic composition according to any one of the preceding claims, comprising from 0.1% to 2.5% of said extract expressed by weight relative to the total weight of the composition.

8. Cosmetic composition according to any one of the preceding claims, for further soothing the skin.

9. Cosmetic composition according to any one of the preceding claims, for increasing the synthesis of collagen.

10. Cosmetic composition according to any one of the preceding claims, for further protecting the skin from free radicals.

11. Use of a cosmetic composition comprising: c) procyanidolic oligomers (PCOs) extracted from pine bark, said oligomers being esterified with at least one fatty acid; and d) a cosmetically acceptable vehicle for repairing the effects of skin aging.

12. Cosmetic treatment method, comprising the application, especially topical, of a cosmetic composition according to any one of the preceding claims.