Methods for treatment of skin and skin appendages with a linseed extract as an active agent activating the synthesis of antimicrobial peptides

Abstract

Methods for the treatment of irritations of the skin and skin appendages caused by microbial stresses are disclosed. The methods include providing a composition comprising a linseed extract, obtained from hydrolysis of linseed proteins, as an active antimicrobial agent in a physiologically acceptable medium, and topically applying the linseed extract onto the skin in need thereof. The methods may be useful in the cosmetic and pharmaceutical field and, more particularly, in the field of dermatology. Cosmetic methods for reinforcing the chemical barrier function of healthy and/or sensitive skin and skin appendages are also disclosed that include the same or similar composition and steps.

Claims

1. A method for the treatment of irritations of the skin and skin appendages caused by microbial stresses by increasing an expression level of defensin and/or cathelicidin antimicrobial peptides without causing a toxic or allergic reaction of the skin, the method comprising: providing a composition comprising a linseed extract, obtained from hydrolysis of linseed proteins, and comprising at least 1.5 to 3.5 g/l of peptide compounds by weight of dry extract, 0.3 g/l or less of sugar by weight of dry extract and includes essentially peptide compounds having a molecular weight below 5 kDa, for pharmaceutical use, as an active antimicrobial agent in a physiologically acceptable medium; and topically applying an effective amount of the linseed extract onto irritated skin of a subject in need twice daily for 24 or 48 hours, thereby increasing the expression level of defensin and/or cathelicidin antimicrobial peptides, and resulting in the treatment of irritations of the skin and skin appendages caused by microbial stresses.

2. The method of claim 1, wherein the method maintains the equilibrium and favorable conditions of the ecosystem of the commensal microflora of the skin, and reinforces the chemical barrier function of sensitive skin and skin appendages, without causing a toxic or allergic reaction of the skin.

3. The method according to claim 1, comprising topically applying an effective amount of the composition to reduce the imbalance of the commensal microbial flora of the skin and skin appendages.

4. The method according to claim 1, wherein the composition includes the linseed extract at a concentration of between 0.05% and about 5%.

5. The method according to claim 1, wherein, after dilution in a physiologically acceptable solvent, the linseed extract contains between 1.5 to 3.5 g/l of peptide compounds by weight of dry extract and 0.3 g/l or less of sugar by weight of dry extract.

Description

(1) FIG. 1 shows, in the form of a histogram, the immunohistochemical detection of the expression level of antimicrobial proteins DEFB1 and LL-37 in the ex vivo skin treated or not treated with 1% linseed extract for 24 h.

EXAMPLE 1

Preparation of Active Principle from Linseed (Linum usitatissimum L.)

(2) In a first step, 1 kg of linseed seeds (Linum usitatissimum L.) is ground in a grain grinder. The flour obtained (1 kg) is placed in the presence of 10 liters of hexane. The mixture is then stirred for 2 hours at room temperature in order to perform a delipidation of the raw material. After filtration and vacuum drying, the powder obtained is suspended in 800 ml of an alkaline aqueous solution ( 1/10 dilution) pH 10 containing 1% polyvinylpolypyrrolidone (Polyclar V ISP). This mixture is stirred for 2 hours at room temperature so as to enable the soluble fractions to be solubilized. After this extraction phase, the medium is clarified by centrifugation, then filtered on a plate. This filtrate, which contains the soluble fractions of the linseed, is then subjected to a protein precipitation by varying the ionic force in a neutral or acid medium, which makes it possible to remove the soluble carbohydrate components, the lipids and the nucleic acids. The medium is brought to pH 3.5. The supernatant is removed and the precipitate is then washed with ethanol, then the solvent is evaporated by vacuum drying.

(3) At this stage, around 50 grams of light yellow powder of raw protein extract are obtained, containing: Proteins: 78% Carbohydrates: 20% Lipids <2%

(4) The protein-rich precipitate is placed in solution in 500 grams of water.

(5) The raw protein extract is then subjected to a series of controlled and selective enzymatic hydrolyses in the presence of 0.5% PVPP (Polyclar V) and cysteine endopeptidases (2 g/l bromelaine and 2 g/l alcalase). After 2 hours of reaction at 50° C. then deactivation of the enzymatic cocktail for 2 hours at 80° C., the hydrolysate is filtered on plates of decreasing porosity, then on a sterilizing cartridge (0.2 μm).

(6) A light-colored hydrolysate is then obtained, measured at 15 to 30 g/l of dry extract, which is then diluted so that the concentration of peptide compounds determined by the Lowry method is between 0.1 and 5 g/l and preferably between 1.5 and 3.5 g/l. The physicochemical analysis of the plant hydrolysate, which constitutes the active principle, shows that its pH is between 4 and 5, and preferably between 4 and 4.5. The linseed extract according to the invention has a dry extract content of between 1 and 8 g/l, and preferably between 0.1 and 5 g/l and includes between 0.1 and 5 g/l of peptide compounds by weight of dry extract and between 0.1 and 2 g/l of sugars by weight of dry extract, preferably the linseed extract according to the invention is diluted so as to contain between 1.5 and 3.5 g/l of peptide compounds by weight of dry extract and between 0.1 and 0.3 g/l of sugars by weight of dry extract.

EXAMPLE 2

Demonstration of the Activating Effect of the Linseed Extract According to Example 1 on the Expression Level of DEFB1 and LL37 Messenger RNA in Keratinocytes

(7) The objective of this study is to determine the influence of the linseed extract according to example 1 on DEFB1 transcript expression and on LL-37 transcript expression. To evaluate the expression level of DEFB1 messenger RNA and the expression level of LL-37 messenger RNA, real-time polymerase chain reaction (PCR) quantifications were performed.

(8) Protocol

(9) NHK cells (normal primary human keratinocytes) are treated with 1% linseed extract according to example 1, twice daily, for 48 hours or are not treated (control).

(10) To evaluate the expression level of DEFB1 messenger RNA and the expression level of LL-37 messenger RNA, it is necessary to isolate the total RNA from the keratinocytes in culture, treated or not treated by the linseed extract according to example 1. The total RNA are then transformed into complementary DNA by the action of a reverse transcriptase enzyme. A quantification of the complementary DNA is then performed by real-time PCR by means of a STEPONEPLUS™ thermocycler (Applied Biosystems). This quantification makes it possible to determine the expression level of DEFB1 messenger RNA and the expression level of LL-37 messenger RNA.

(11) Results

(12) An increase by 31% of the expression level of DEFB1 messenger RNA and an increase by 23% of the expression level of LL-37 messenger RNA is observed in the cells after 48 hours of treatment with the linseed extract according to example 1, by comparison with the untreated cells.

(13) Conclusion

(14) The linseed extract according to example 1 increases the expression level of messenger RNA encoding DEFB1 and the expression level of messenger RNA encoding LL-37, in normal human keratinocytes.

EXAMPLE 3

Demonstration of the Activating Effect of the Linseed Extract According to Example 1 on the Expression of the Antimicrobial Protein DEFB1 and on the Expression of the Antimicrobial Protein LL-37 in Keratinocytes

(15) The objective of this study is to determine the influence of the linseed extract according to example 1 on the expression of the antimicrobial protein DEFB1 and the expression of the antimicrobial protein LL-37. For this, the expression level of the DEFB1 protein and the expression level of the LL-37 protein were evaluated by immunocytochemistry on keratinocytes treated for 24 hours with the 1% linseed extract according to example 1 or without treatment (control) and embedded in paraffin.

(16) Protocol

(17) NHK cells (normal primary human keratinocytes) cultivated. These cells are then fixed with 10% formol, then embedded in paraffin. The cell block is then cut into sections with a thickness of 4 μm with a microtome knife and are then transferred onto a slide.

(18) The sections are removed from the paraffin with 100% xylene, then rehydrated in successive alcohol baths: 2 100% ethanol baths (EtOH) for 2 minutes, 1 95% EtOH bath for 2 minutes, 1 90% EtOH bath for 2 minutes and 1 H.sub.2O bath for 5 minutes. The slides are submerged in a citric acid buffer at 0.01 M pH 6 and heated to a light boil in order to facilitate access of the antibody to the protein of interest. The sections are incubated with 5% BSA for 30 minutes, then with the primary antibody: an anti-“DEFB1” polyclonal rabbit antibody ab14425 (Abcam, Cambridge, UK), diluted to 1/500 in PBS or an anti-“LL-37” monoclonal mouse antibody sc-166770 (Tebu Santa Cruz, Calif., USA) diluted to 1/75 in PBS, for 1½ hours while stirring and at room temperature. After a plurality of washings with PBS, the sections are incubated with the secondary fluorescent antibody (anti-rabbit Antibody Alexa Fluor 488 A21206 (Invitrogen, Fisher)) diluted to 1/1000 in PBS for 1 hour at room temperature. The cell nuclei are marked with 0.3 μM 4′6′-diamidino-2-phenylindole (DAPI) (Molecular Probes). The sections are rinsed for 5 minutes in PBS. The expression of the antimicrobial protein DEFB1 and the antimicrobial protein LL-37 is detected by means of a fluorescence microscope (objective 40×).

(19) Results

(20) The results show that there is an increase by 118% of the expression of the antimicrobial protein DEFB1 and an increase by 136% of the expression of the antimicrobial protein LL-37 in keratinocytes treated with the 1% linseed extract according to example 1 after 24 hours, by comparison with untreated keratinocytes.

(21) Conclusion

(22) The linseed extract according to example 1 stimulates the expression of the antimicrobial protein DEFB1 and the expression of the antimicrobial protein LL-37 in keratinocytes.

EXAMPLE 4

Demonstration of the Activating Effect of the Linseed Extract According to Example 1 on the Expression of the Antimicrobial Protein DEFB1 and on the Expression of the Antimicrobial Protein LL-37 in Ex Vivo Skin

(23) The objective of this study is to determine the influence of the linseed extract according to example 1 on the expression of DEFB1 and on the expression of LL-37. To evaluate the expression level of DEFB1 and LL-37, an immunolabeling of DEFB1 and LL-37 on ex vivo skin sections was performed.

(24) Protocol

(25) Human skin biopsies are kept in culture ex vivo, then treated, twice daily for 24 hours, by topical application of 20 μl of a 1% solution of the linseed extract according to example 1 or not treated.

(26) The skin biopsies are then fixed, then embedded in paraffin after passage through a Shandon Hypercenter XP automatic apparatus (Shandon, UK). The skin biopsies embedded in paraffin are then cut into sections having a thickness of 4 μm with a microtome knife, which are themselves transferred to a slide. The sections are removed from the paraffin with 100% xylene, then rehydrated in successive alcohol baths: 2 100% ethanol baths (EtOH) for 2 minutes, 1 95% EtOH bath for 2 minutes, 1 90% EtOH bath for 2 minutes and 1 H.sub.2O bath for 5 minutes. The slides are submerged in a citric acid buffer at 0.01 M pH 6 and heated to a light boil in order to facilitate access of the antibody to the protein of interest. The sections are incubated with 5% BSA for 30 minutes, then with the primary antibody: an anti-“DEFB1” polyclonal rabbit antibody ab14425 (Abcam, Cambridge, UK), diluted to 1/500 in PBS or an anti-“LL-37” monoclonal mouse antibody sc-166770 (Tebu Santa Cruz, Calif., USA) diluted to 1/100 in PBS, for 1½ hours while stirring and at room temperature. After a plurality of washings with PBS, the sections are incubated with the secondary fluorescent antibody (anti-rabbit Antibody Alexa Fluor 488 A21206 (Invitrogen, Fisher)) diluted to 1/1000 in PBS for 1 hour at room temperature. The cell nuclei are marked with 0.3 μM 4′6′-diamidino-2-phenylindole (DAPI) (Molecular Probes). The sections are rinsed for 5 minutes in PBS. The expression of the antimicrobial protein DEFB1 and the antimicrobial protein LL-37 is detected by means of a fluorescence microscope (objective 40×).

(27) Results

(28) The set of results is presented in FIG. 1.

(29) The evaluation of the fluorescence obtained on the sections by immunohistochemistry shows that the skin biopsies treated with the linseed extract according to example 1 have a highly significantly significant (+240%) higher level of expression of DEFB1 and (+140%) of the expression of LL-37 by comparison with untreated skin biopsies.

(30) Conclusion

(31) A positive effect on the expression of the antimicrobial protein DEFB1 and on the expression of the antimicrobial protein LL-37 is obtained as a result of the treatment with the linseed extract according to example 1 on human skin biopsies.

EXAMPLE 5

Demonstration of the Effect of the Linseed Extract According to Example 1 on the Number and Secreting Activity of Lamellar Bodies in Ex Vivo Skin

(32) The objective of this study is to determine the influence of the linseed extract according to example 1 on the number of lamellar bodies, the antimicrobial protein storage site, present at the interface of the stratum granulosum and stratum corneum. To observe lamellar bodies, electron microscopy images obtained from an ex vivo skin section were produced.

(33) Protocol

(34) Human skin biopsies are kept in culture ex vivo, then treated, once daily for 24 hours, by topical application of 20 μl of a 1% solution of the linseed extract according to example 1 or not treated.

(35) The skin biopsies are then fixed by means of a Karnovsky fixative buffer (Electron Microscopy Sciences, Hatfield, UK) for 1 hour at room temperature, then overnight at 4° C. After having been rinsed by a Sodium Cacodylate buffer at 0.1 M (Sigma, Steinheim, Germany), the biopsies are post-fixed in 1% Osmium tetroxide OsO4 for 1 hour, then rinsed and dehydrated in successive alcohol baths. The samples are infiltrated and included in a low-viscosity Epon-Epoxy mixture.

(36) The sections are produced by means of a microtome knife equipped with a diamond blade. The sections are then colored with uranyl acetate and lead citrate. The observation is performed with a transmission electron microscope at 60 keV.

(37) Results

(38) The observation of the electron microscopy images shows that the skin biopsies treated with the linseed extract according to example 1 have a larger number of lamellar bodies at the interface of the stratum granulosum and stratum corneum by comparison with untreated skin biopsies.

(39) Conclusion

(40) A positive effect on the number of lamellar bodies, the antimicrobial peptide storage site, is observed as a result of the treatment with the linseed extract according to example 1 on human skin biopsies.

EXAMPLE 6

Demonstration of the Antimicrobial Effect Produced by the Linseed Extract According to Example 1 in Keratinocytes on the Bacterial Growth of Staphylococcus aureus

(41) The objective of this study is to determine the effect of increased synthesis of antimicrobial proteins produced by keratinocytes as a result of stimulation by the linseed extract according to example 1 on the bacterial growth of Staphylococcus aureus. For this, a radial diffusion test was performed.

(42) Protocol

(43) NHK cells (normal primary human keratinocytes) are treated with the linseed extract according to example 1, twice daily, for 24 hours, or are not treated (control). To produce a negative control, NHK culture medium not having had any contact with the cells is used. The supernatant of the cells is then recovered and 150 μl are used to soak a disk having a 6-mm diameter. The disks are then placed on a S. aureus culture seeded in the mass (4 disks for each condition and Petri dish). After 48 hours, a radial diffusion area is observed. Photographs of the inhibition areas are taken with the QImaging Micropublisher 3.3 RTV camera and their diameters are calculated by the Q-CAPTURE PRO7™ software program.

(44) Results

(45) The results show that there is a 19% increase in the diameter of the bacterial growth inhibition area after application of supernatant resulting from the culture of keratinocytes treated with the 1% linseed extract according to example 1 after 24 hours, by comparison with untreated keratinocytes.

(46) Conclusion

(47) The linseed extract according to example 1 enables inhibition of bacterial growth.

EXAMPLE 7

Demonstration of the Antimicrobial Effect Produced by the Linseed Extract According to Example 1 in Keratinocytes on the Bacterial Growth of Staphylococcus aureus

(48) The objective of this study is to determine the effect of increased synthesis of antimicrobial proteins produced by keratinocytes as a result of stimulation by the linseed extract according to example 1 on the bacterial growth of Staphylococcus aureus. For this, a bacterial growth inhibition test was performed.

(49) Protocol

(50) NHK cells (normal primary human keratinocytes) are treated with the linseed extract according to example 1, twice daily, for 48 hours, or are not treated (control). To produce a negative control, NHK culture medium not having had any contact with the cells is used. The supernatant of the cells is then recovered and 200 μl are used and contaminated by a solution of S. aureus. This supernatant/S. aureus mixture is incubated for 3 hours at 37° C. then spread on TSA agar dishes. After 48 hours, S. aureus colonies have developed and are visible to the naked eye. Photographs of the dishes are taken with the QImaging Micropublisher 3.3 RTV.

(51) Results

(52) The results show that there is a 79% decrease in the number of S. aureus colonies developed under the condition in which the supernatant was obtained from a culture of keratinocytes treated with the 1% linseed extract according to example 1 after 24 hours, by comparison with untreated keratinocytes.

(53) Conclusion

(54) The linseed extract according to example 1 enables a reduction in the number of bacterial colonies.

EXAMPLES

Preparation of Compositions

Protective Calming Day Cream

(55) TABLE-US-00001 Brand names INCI names Weight % Phase A EMULIUM ® Cetyl alcohol (and) Glyceryl Stearate 4.00 Delta (and) PEG-75 Stearate (and) Ceteth-20 (and) Steareth-20 LANETTE ® O Cetearyl Alcohol 1.50 D C 200 Dimethicone 1.00 Fluid/100 cs DUB 810C Coco Caprylate/Caprate 1.00 DPPG Propylene Glycol Dipelargonate 3.00 DUB DPHCC Dipentaerythrityl 1.50 Hexacaprylate/Hexacaprate CEGESOFT ® PS6 Vegetable Oil 1.00 Vitamin E Tocopherol 0.30 Phase B Demineralized Aqua qsf 100 water Glycerin Glycerin 2.00 CARBOPOL ® Acrylates/C10-30 Alkyl Acrylate 0.15 EDT 2020 Crosspolymer KELTROL ® BT Xanthan Gum 0.30 Allantoin Allantoin 0.5 Phase C Sodium Sodium Hydroxide 0.30 Hydroxide (10% sol.) Phase D Demineralized Aqua 5.00 water STAY-C ® 50 Sodium Ascorbyl Phosphate 0.50 Phase E Butylene Glycol Butylene Glycol 2.00 ROKONSOL Phenoxyethanol (and) Methylparaben 1 MEP (and) Ethylparaben (and) Propylparaben Dekaben CP Chlorphenesin 0.20 Phase F GP4G Water (and) Artemia Extract 1.00 Linseed extract 1.5 according to example 1

(56) Prepare phase A and heat to 75° C. Prepare phase B by dispersing carbopol, then xanthan gum while stirring. Let rest until perfect homogeneity is obtained. Heat B to 75° C.

(57) At 75° C., emulsify A in B under rotor-stator stirring. Neutralize with phase C under rapid stirring. After cooling at 40° C., add limpid phase D, then phase E (preheated to 40° C. and homogenized until perfect limpidity is obtained). The cooling is continued with light stirring until 25° C. and phase F is added.

(58) 2—Body Milk

(59) TABLE-US-00002 Brand names INCI names Weight % PHASE A MONTANOV ® C14-22 Alcohols (and) C12-20 Alkyl 3.00 L Glucoside Waglinol 2559 Cetearyl Isononanoate 4.00 TEGOSOFT ® C12-15 Alkyl Benzoate 3.00 TN Apricot Seed Prunus Armeniaca (Apricot) Kernel Oil 2.00 Oil Avocado Oil Persea Gratissima (Avocado) Oil 1.00 ABIL ® 350 Dimethicone 1.00 PHASE B Demineralized Aqua (Water) Qsf water 100 Allantoin Allantoin 0.5 PHASE C SIMULGEL ® EG Sodium Acrylate/Acryloyldimethyl 0.4 Taurate Copolymer (and) Isohexadecane (and) Polysorbate 80 Copolymer (and) Polysorbate 80 PHASE D ROKONSAL MEP Phenoxyethanol (and) Methylparaben 1 (and) Ethylparaben (and) Propylparaben GERMALL ® 115 Imidazolidinyl Urea 0.20 PHASE E Linseed extract 1 according to example 1

(60) The constituents of phase A and phase B are heated separately between 70° C. and 75° C. Phase A is emulsified in phase B while stirring. Phase C is added, at 45° C., with increased stirring. Phases D and E are then added when the temperature is below 40° C. The cooling is continued until 25° C. under a heavy stirring.