FR2853835A1 - Lipid-containing extracts from fruits low in lipids are useful for the topical treatment of dermal inflammations and protect the skin against physical or chemical damage by restoring the epidermal barrier - Google Patents

Abstract

Extracts from the pulp of fruits low in lipids, comprising less than 10% by weight of triglycerides and 5 - 50% by weight of polar lipids, are useful for preparing a topical treatment of dermal inflammations. ACTIVITY : Dermatological; Antiinflammatory. No suitable biological data provided. MECHANISM OF ACTION : Intracellular Adhesion Molecule-1 (ICAM1) and Interleukin-1a (IL1a) inhibitors.

Description

The present invention relates to an extract of fruit pulp

  poor in lipids, its use as a medicament or as a cosmetic agent, in particular for restoring the epidermal barrier function, of the compositions containing it, as well as its method of preparation.

  Fruits that are naturally rich in lipids are used in cosmetology or pharmacology. Serenoa repens oil is used in anti-seborrheic products or against alopecia for its 10 inhibitory activities of 5 alpha reductase. FR2764804 describes the preparation of a lipid extract of the fruit of the Safoutier tree which can be used for the care of the skin, mucous membranes and / or integuments.

  The kernels of certain fruits have also been valued in cosmetology or pharmacology as a source of lipids. FR2780277 describes a process for the preparation of a lipid extract obtained from mirabelle fruit kernels and its use for the care of the skin, lips, mucous membranes and / or integuments. FR 2553788 describes a process for the preparation of a lipid extract obtained from the almonds of the argan fruit and its use for restoring the elasticity and suppleness of dry skin.

  On the other hand, fruits whose pulp is low in lipids have so far never aroused any particular interest as a source of lipids. As a result, they have never been exploited for the lipids they contain.

  In the context of the present invention, the term “fruit low in lipids” is understood to mean a fruit whose pulp contains less than 10% by weight of lipids.

  However, the inventor remarked very surprisingly that from fruit pulp poor in lipids, one can obtain an extract which has very interesting properties, making it usable in cosmetic compositions and! or pharmaceuticals.

  The subject of the present invention is an extract comprising less than 10% by weight of triglycerides and from 5 to 50% by weight of polar lipids, the percentages being expressed relative to the total dry matter of the extract, said extract being obtained by extraction from the pulp of fruits low in lipids.

  Polar lipids are defined as the lipids which precipitate with acetone.

  This extract has the property of restoring the barrier function of the skin and can therefore be used in compositions as an anti-inflammatory agent or as an agent for protecting the skin from physical or chemical attack.

  Advantageously, the fruits low in lipids from which the extract object of the present invention is prepared are chosen from apricot, acerola, cranberry, pineapple, chokeberry, banana, blackcurrant, cherry, lemon, cranberry, dog rose, fig, strawberry, raspberry, morello cherry, redcurrant, hibiscus, kiwi, tangerine, blackberry, blueberry, orange, 15 grapefruit, passion, papaya, peach, pear, apple, plum, sloe, grape, mountain ash or elderberry, as well as their mixtures.

  Mixture means the mixture of at least two fruits.

  Preferably, the extracted fruits are chosen from lemon, wild rose, raspberry, morello cherry, red currant, tangerine, blueberry, orange, as well as their mixtures.

  The part consisting of polar lipids comprises phospholipids, sterols, glycosterols, ceramides, cerebrosides and glycolipids.

  Advantageously, the phospholipids represent 0.5 to 30% by weight relative to the total dry matter of the extract.

  Advantageously, the phospholipids include phosphoethanolamines, phosphatidylinositol, lysophosphatidylethanolamines, phosphatidic acids, phosphatidylcholine and lysophosphatidylcholines.

  Advantageously, the ceramides represent 0.1 to 10% by weight relative to the total dry matter of the extract.

  The present invention also relates to the process for preparing the extract comprising the steps consisting in: - separating the fruit pulp from its seeds or from its core, - drying the fruit pulp thus obtained so as to obtain a content of water less than about 10% by weight, carry out the extraction of the dried pulp using a solvent of intermediate polarity, with a solvent / dried pulp mass ratio of between about 4/1 and about 20/1, and at a temperature between room temperature and boiling temperature, carry out a liquid / solid separation so as to collect an extraction solution, - evaporate the solvent from said extraction solution to obtain said extract.

  Thus, according to the process which is the subject of the present invention, the fruits are first of all stripped of their kernel or of their seed, then the pulp thus obtained is dried.

  The fruit pulp is dried so as to obtain a water content of less than about 10% by weight, preferably less than 5% by weight.

  The lower the water content, the better the extraction yield of the polar lipids.

  Advantageously, the drying of the pulp is carried out by lyophilization, atomization or on rollers so as to obtain a fruit powder or flakes. The drying can also be carried out on supports such as lactose or maltodextrin.

  Once dried, the fruit pulp is extracted with solvents of intermediate polarity with a solvent / pulp mass ratio of between about 4/1 and 20/1 and at a temperature between room temperature and the boiling temperature.

  In the polarity scale, the solvents of intermediate polarity are between methanol and ethyl acetate. Advantageously, the solvent of intermediate polarity is chosen from an alcohol comprising 1 to 4 carbon atoms, a ketone, a water / alcohol mixture comprising 1 to 4 carbon atoms and a water / ketone mixture. Preferably, the solvent chosen is an alcohol such as ethanol or isopropanol.

  Advantageously, the extraction is carried out with stirring or statically for a period of between 1 and 24 hours approximately.

  Once the extraction has been carried out, a solid / liquid separation is carried out so as to collect an extraction solution. The extraction and separation steps can be carried out several times in succession, preferably 2 times, and the extraction solutions thus obtained are then combined.

  The extraction and separation steps can be carried out in a single step as in the case of leaching.

  The solvent is then evaporated from said extraction solution thus obtained to obtain a lipid extract.

  Advantageously, the solvent of said extraction solution is evaporated, completely or partially, under reduced pressure and at the boiling point of the solvent under this pressure.

  In the case of complete evaporation, a dry lipid extract is obtained. In the case of partial evaporation, a pasty or liquid lipid extract is obtained containing 0.1% to 20% by weight of dry matter.

  The present invention also relates to the compositions comprising the extract. Preferably, these compositions are cosmetic or pharmaceutical and for topical use.

  Advantageously, the compositions according to the present invention contain 0.0001 to 1% by weight of dry matter of the lipid extract.

  The dry, pasty or liquid extract can be dissolved in a cosmetically and or pharmaceutically acceptable solvent, in particular an oil, butylene glycol or propylene glycol.

  The extract can also be present in the pharmaceutical compositions according to the present invention in the form of liposomes. Advantageously, these liposomes are formed spontaneously in aqueous solution when water is added during or before the complete evaporation of the solvent from the extraction solution of the process which is the subject of the present invention.

  Another subject of the present invention is the use of the extract as a medicament or as a cosmetic agent, in particular for restoring the epidermal barrier function. In particular, this extract can be used as an anti-inflammatory agent or as an agent for protecting the skin from physical or chemical attacks.

  The skin is the organ most exposed to environmental damage. The structural complexity of the skin tissue gives it 10 various functions: the hypodermis acts as a thermal insulator, the dermis contributes to resistance and elasticity, while the covering epithelium, stratified and cornified, is responsible for the sealing therefore of the barrier function. The stratum cornea, the most superficial part of the epidermis, is made up of non-living cells, fulfilling protective functions, regulating the differentiation and multiplication of kerinocytes. Consequently, it plays a key role in the control of skin homeostasis. Indeed, the stratum corneum prevents water losses and opposes physical and chemical aggression as well as the penetration of microorganisms.

  During an acute or subacute disturbance of the skin by surfactants, organic solvents or mechanical alteration, a recovery phase of the barrier lasting several hours takes place. The kinetics of repair, which varies from subject to subject, depends on the subject's age, the intensity and the extent of the lesion. This recovery is directly related to a series of metabolic responses including protein and nucleic lipid biosyntheses. These various metabolic responses are set up following biological signals which accompany the breakdown of the barrier function. Among the cascade of molecular and cellular events occurring during an alteration of the stratum corneum, there is an increase in keratinocytic cytokines, such as ILla and inter keratinocytic adhesion molecules such as ICAM1 (Intra Cellular Adhesion Molecule-1) epidermal and dermal compartments of the skin system (Nickoloff BJ, Naidu Y, Perturbation of epidermal barrier function correlates with initiation of cytokine cascade in human skin. J. Am Acad Dermatol. 1994 Apr; 5 30 (4): 535-546 - Elias PM, Wood LC, Feingold KR. Epidermal pathogenesis of inflammatory dermatoses. AM. Contact Dermat., 1999 Sept; 10 (3) = 119126 - Elias PM, Feingold KR ,. Coordinate regulation of Epidermal Differentiation and barrier Homeostasis. Skin Pharmacol Appl Skin Physiol 2001; 14 (suppl 1): 28-34).

  These molecular events set up during physical and / or chemical stress of the stratum cornea precede any movement of inflammatory cells from the circulation to the dermis and epidermis, thus inducing an inflammatory skin focus. The horny layer of the epidermis is therefore a biosensor regulating cutaneous homeostasis, following exogenous stresses. Tests which are developed later in the description have demonstrated the restoration of the barrier function by topical application of a lipid fruit extract according to the present invention thus preventing the establishment of the inflammatory markers ICAM and ILla, following a chemical attack inducing cellular metabolic disturbances. 20 Examples of a process for preparing a lipid extract of fruit low in glycerides:

Example 1

  100 kg of dried raspberry flakes are extracted with 700 l of isopropanol at reflux for 2 h and with stirring. After returning to ambient temperature, the fruits extracted from the extraction solution are separated by filtration. The collected solution is concentrated. The dry matter is determined. Then a quantity of water 50 times greater than the dry matter is added. Then reconcentrated until a liposomal aqueous solution is obtained whose dry matter is 5% by weight. The extract obtained is analyzed by thin layer chromatography in order to identify the molecules present.

  Thus, phospholipids, ceramides, cerebrosides, sterols, glycolsysterols and glycolipids are identified. Phospholipids and, ceramides and triglycerides are also assayed. The amount of total polar lipids is 45% by weight relative to the total dry matter. The total phospholipid content is 25% by weight relative to the total dry matter. The total ceramide content of 3.5% by weight relative to the total dry matter. The triglyceride content is 5% by weight relative to the total dry matter.

Example 2

  500 kg of a mixture of dried currants, raspberries and blueberries (1/3, 1/3, 1/3) is extracted with 5 tonnes of ethanol at 500C for 5 hours with stirring at room temperature. The extraction residues from the solution are filtered off. The latter is completely evaporated, until a pasty extract is obtained.

  The thin layer chromatographic analysis makes it possible to identify the following molecules: phospholipids, ceramides, cerebrosides, sterols, glycosterols and glycolipids. The content of total polar lipids is 15% by weight relative to the total dry matter. The total phospholipids are dosed at 11% by weight relative to the total dry matter and the ceramides at 2% by weight relative to the total dry matter. The triglyceride content is 10% by weight relative to the total dry matter.

Example 3

  kg of a mixture of dried lemon, tangerine and orange (1/3, 1/3, 1/3) are extracted with 200 kg of isopropanol at room temperature statistically by leaching. The solution obtained is concentrated to 2 kg. The dry matter content of the concentrate is determined and 50 times more butylene glycol is added to the dry matter. The rest of the volatile solvent is evaporated. There is therefore obtained an extract of butylene glycol containing 2% by weight of dry matter. The molecules present in the extract are checked by thin layer chromatography. Phospholipids, ceramides, cerebrosides, sterols, glycosylsterols and glycolipids are identified.

  The polar lipids are determined at 20% by weight relative to the weight of total dry matter, the phospholipids at 16% by weight relative to the total dry matter and the ceramides at 1.5% by weight relative to the total dry matter . The triglyceride content is 11% by weight relative to the total dry matter.

  Evaluation of the restoration of the epidermal barrier function by topical application of a lipid extract of fruit low in lipids.

  The tests are carried out on the skin explant coming from mammary plasties maintained in survival in an adequate medium.

  The tests are carried out on a skin explant serving as an absolute control, on an explant defatted by an ether / acetone mixture then stressed by a chemical stress induced by topical application of a solution of lauryl sulfate or SDS (Sodium Dodecyl Sulfate) and on a defatted skin, treated with the fruit extract prepared according to Example 1, at a rate of 500 pJg / ml and treated with a chemical stressor.

  The test protocol is described by Williams ML, Elias PM, From besket weave to barrier. Arch Dermatol 1993; 129: 626-629.

  Samples of skin explants having undergone these preparations 25 are taken in order to study the various parameters proposed: - Control of the viability and metabolic activity of the cells using the MiT test (ref. Mosmann Tim. 1993 - Journal of immunological methods 65: 55-63).

  - Detection of an inflammatory cytosoluble signal: immunostaining of the ICAMi protein.

  - Detection of an inflammatory membrane signal: whatever the marker to be revealed, the principle of the immunostaining technique is the same, a special immunostaining kit is used.

  On cryocuts attached to acetone, the non-specific sites are saturated using a 3% BSA solution.

  The primary antibody recognizing the protein to be identified is applied to the section, the samples are then rinsed using phosphate buffer (PBS).

  The biomarked secondary antibody directed against the species which produced the protein of interest is then applied. The sections are again rinsed with PBS and brought into contact with the streptavidin peroxidase protein coupled to an enzymatic marker.

  This enzyme is then visualized by a specific chromogenic substrate DAB. The sections are then mounted on the Gel / Mount and observed 24 hours later in white light.

  Observation with the right light microscope in white light and with the confocal leads to the following results.

  Evaluation of the viability of explants after metabolism of MTT.

  The absolute control leads to a strong metabolism of MTT, resulting in the presence of blue-to-blue formazan crystals in the epidermis and in the dermis.

  Chemical aggression by the SDS, induced on the defatted control, a decrease in the metabolism of MIT in the epidermis.

  For the explant treated with fruit lipids, we observe a more intense and denser metabolism of MUI than that noted at the level of the stressed delipidated control which revealed an alteration of the barrier. The return to normal by the treatment with fruit lipids indicates a protection and a reconstitution of the skin barrier by these same fruit lipids.

  Study of the expression of the inflammatory cytokine IL The absolute control shows an expression of the IL on the entire epidermis with more sustained labeling at the level of the basal layer.

  Compared to the absolute control, the stressed delipidated control has an overexpression of Ila in the basal and suprabasal keratinocytes.

  By the treatment with fruit lipids, the intensity of labeling is very discreet and in certain areas of the basal base the labeling is less sustained than that noted in the absolute control.

  Study of the expression of the ICAMi protein The absolute control has a marking which is essentially localized at the level of the cells of the upper dermis close to the epidermal dermal junction.

  The chemical aggression induced an over-expression of ICAMi on the delipidated control in the basal keratinocytes and the dermal cells, endothelial cells, dermal dendritic cells, macrophages, etc.

  With regard to the skin treated with the fruit lipids, we observe a location and a marking identical to those noted in the absolute control.

  Immunostaining results table | 1 ILla | ICAM1I Absolute control ++ + Stressed defatted skin +++ ++ Stressed defatted skin extract of prepared fruit +/- +

according to example 1

  (500 pg / ml) II The interruption of the barrier function by superficial defatting of the skin explant using an organic solvent favored the induction of pro inflammatory molecular and cellular events following exogenous chemical stress. : in fact, the inflammatory epidermal cytokine ILla and the intercellular adhesion molecule ICAMi are overexpressed during a deteriorated barrier function. The metabolic activity necessary for restoring the barrier function is greatly reduced, due to chemical stress.

  The application of a lipid extract of fruits according to the present invention has contributed to restoring the barrier function inhibiting the pro-inflammatory effect and the tissue metabolic alteration induced by chemical or mechanical stress: skin homeostasis is thus found thanks to topical application of the lipid extract of fruits according to the present invention. 15 Evaluation of the Effect of a Lipid Extract of Fruit Low in Glycerides on the Energy Metabolism of the Skin This study aims to evaluate the energizing efficiency of a lipid extract of fruit low in glycerides prepared according to the Example 1 by phosphorus 31 magnetic resonance spectroscopy (SRM31P).

  The study was carried out on a group of 15 women with dehydrated skin. The lipid fruit extract was applied twice a day for 28 days, its activity was compared to some of the untreated skin. The results obtained at the different times were compared with those obtained at To (treated area / control area).

  The variations in the energy metabolism of the skin were determined by magnetic resonance spectroscopy of Phosphorus 31 by measuring the relative concentrations of the main phosphorylated metabolites 30 of the skin: Inorganic Phosphate (Pi), Phosphocreatine (Pcr), Adenosine Tri-phosphate (ATP).

  The spectroscopic acquisitions were carried out with an NMR imaging spectrometer equipped with a Proton-Phospore dual-tuned surface antenna developed for in vivo magnetic resonance spectroscopy of the skin.

  No significant increase is observed 4 hours or 7 hours after the application of the product PO 1662. of the lipid extract of fruit.

  On the other hand, after 28 days of application of the product PO 1662 of the lipid extract of fruit, an increase in the Pcr / Pi ratios of + 8%, ATP / Pi of + 8.5% and total Pcr / P of + 3 , 1% as well as the phosphorylation index of + 1.5% was highlighted significantly.

  ATP molecules provide the energy needed by cells and tissues. Phosphocreatine is a molecule which constitutes an immediately available energy reserve, responsible for reconstituting the ATP reserves consumed during a period of cellular ischemia by supplying a phosphate group to the ADP molecules.

  The results obtained after treatment with the lipid extract of fruit indicate that the significant increase in the Pcr / Pi ratio (+ 8%) after 28 days of application is mainly due to an increase in phosphocreatine since the Pcr / Ptotal ratio increases also significantly (+ 3.1%).

  In addition, for the Pcr / Pi ratio, a significant difference is observed after 28 days of treatment between the treated arms and the control arms.

  The results indicate an increase in the ATP level which is demonstrated by the significant increase at T + 28 days in the ATP / Pi 25 ratio (+ 8.5%).

  In addition, the increase in the ATP pool is confirmed by the significant increase in the phosphorylation index (+ 1.5%) which is a reflection of the state of energy metabolism.

  This study by SRM31P therefore demonstrates an improvement in the energy status of the skin after treatment with the lipid extract of fruit. This effect can be attributed to the product since no significant variation was observed on the control forearm throughout the duration of the study.

  Examples of the compositions containing a lipid extract of fruit low in lipids The following compositions were produced from the lipid extract of fruit poor in glycerides obtained according to Example 1.

  Osmo-active treatment (I / O) Ingredients Quantity in grams Lipid extract 0.001 to 1 AND 0.4 Sodium hyaluronate 0.001 Tocopheryl acetate 0.2 Cyclomethicone / PEG / PPG-18/18 20 Dimethicone Glycerin 19 Butylene glycol 8 PEG 4 10 Hydroxypropylcellulose 0.1 Sodium chloride 0.5 Phenoxyethanol 0.35 Blue acid 3 0.00035 Yellow 5 0.00005 Fragrance 0.2 Water qs 100 Gel Ingredients Quantity in grams Lipid extract 0.001 to 1 Alpha tocopheryl acetate 0.2 Magnesium Ascorbyl phosphate 0.001 Squalane 4.5 Cyclomethicone 8 Sucrose stearate 1.5 Glycerin 4 PEG- 32 4 Methyl Gluceth-20 02.5 Triethanolamine 0.35 Methyl sodium paraben 0.2 Phenonip 0.5 Fragrance 0.25 Water qsq 100 Rich care Ingredients Quantity in grams Lipid extract 0.001 to 1 Amonium Glycyrrhizate 0.05 Cetearyl alcohol / cetearyl 2 sodium sulphate Sucrose stearate 4 Glyceryl stearate 2.6 Glyceryl stearate Cocos nucifera oil (5.8 coconut) Meneral oil 6.5 Propylene glycol 3.2 dicaprylate / dicapr ate Fruit Butyrospermum parkii (shea butter 4.2) Cetaryl ethylhexanoate / Isopropyl 5 myristate Cyclomethicone / Dimethiconol 3 Glycerin 2.5 Butylene glycol 2.5 C13-14 Isoparaffin / Laureth- 0.5 7 / Polyacrylamide Citric acid 0.136 Paraben propyl 0.4 Benzyl alcohol 0.4 Yellow 0.4 0.0018 Yellow 6 0.005 Water qs 100 Microemulsion Ingredients Quantity in gram Lipid extract 0.001 to 1 Alpha tocopheryl acetate 0.2 Decaglycerol mono myristate 12 Dicapryl carbonate 3 Squalane 5 Octyldecanol 3 Polydecene 3 Capric / caprylic triglyceride 35.15 Decamethyl cyclopentasiloxane 17 Phenoxyethanol 0.6 Phenoxyethanol 0.6 Phenoxyethanol

Claims (12)

  1- Extract comprising less than 10% by weight of triglycerides and from 5 to 50% by weight of polar lipids, the percentages being expressed relative to the total dry matter of the extract, said extract being obtained by extraction from fruit pulp low in lipids.   2- Extract according to claim 1 wherein the polar lipids contain 0.5 to 30% by weight of phospholipids relative to the total dry matter of the extract.   3- Extract according to claim 1 wherein the polar lipids contain 0.1 to 10% by weight of ceramides relative to the total material of the extract.   4- Extract according to any one of claims 1 to 3 characterized in that said fruit low in lipids is chosen from apricot, acerola, cranberry, pineapple, chokeberry, banana, blackcurrant , cherry, lemon, cranberry, dog rose, fig, strawberry, raspberry, morello cherry, redcurrant, hibiscus, kiwi, tangerine, blackberry, blueberry, orange, grapefruit, passion, papaya, peach, pear, apple, plum, sloe, grape, mountain ash or elderberry, as well as their mixtures.   5- Composition containing 0.0001 to 1% by weight of dry matter of the extract according to any one of claims 1 to 4.   6- Composition according to claim 5 characterized in that said extract is dissolved in a cosmetically and / or pharmaceutically acceptable solvent, in particular an oil, butylene glycol or propylene glycol.   7- Composition according to claim 5 characterized in that said extract is present in said composition in the form of liposomes.   8- Extract according to any one of claims 1 to 4 for its use as a medicament or as a cosmetic agent.   9- Extract according to claim 8 for restoring the epidermal barrier function.   1 0-extract according to claim 8 for use as an anti-inflammatory agent or agent for protecting the skin from physical or chemical attack.   11-A method of preparing the extract according to any one of claims 1 to 4 comprising the steps of: - separating the fruit pulp from its seeds or its core, - drying the fruit pulp thus obtained so as to obtain a water content of less than approximately 10% by weight, 20 - carry out the extraction of the dried pulp using a solvent of intermediate polarity, with a solvent / dried pulp mass ratio of between approximately 4/1 and approximately 20 / 1, and at a temperature between room temperature and the boiling temperature, carry out a liquid / solid separation so as to collect an extraction solution, - evaporate the solvent from said extraction solution to obtain said extract.   12-A method according to claim 11 characterized in that the fruit pulp is dried so as to obtain a water content preferably less than 5% by weight.   13-A method according to any one of claims 1 1 and 12 characterized in that said solvent of intermediate polarity is chosen from an alcohol comprising 1 to 4 carbon atoms, a ketone, a water / alcohol mixture comprising 1 to 4 atoms of carbon and an eaulketone mixture.