NOVEL COSMETIC AND DERMATOLOGICAL USES OF AN EXTRACT OF CISTUS MONSPELIENSIS

Abstract

Novel cosmetic and dermatological uses of an extract of Cistus monspeliensis The invention relates to the non-therapeutic cosmetic use of an extract of Cistus monspeliensis for increasing and/or maintaining tissue homeostasis in the skin and/or the mucous membranes and/or the skin appendages, by increasing and/or maintaining the barrier function and/or cell differentiation in the skin and/or the mucous membranes and/or the skin appendages. Another subject relates to a non-therapeutic cosmetic care process notably comprising the topical application of the extract of C. monspeliensis or of a cosmetic composition comprising same. Yet another subject relates to the extract of C. monspeliensis, for its use for preventing and/or treating diseases induced by a decrease in the gene and/or protein expression of ECM1, advantageously lichen sclerosus or lipoid proteinosis, and/or for preventing and/or treating scars.

Claims

1.-17. (canceled)

18. A cosmetic method for increasing and/or maintaining tissue homeostasis in the skin and/or the mucous membranes and/or the skin appendages comprising administering an effective amount of an extract of the aerial parts of Cistus monspeliensis or of a cosmetic composition containing it to an individual.

19. The cosmetic method according to claim 18, wherein the method increases and/or maintains the gene and/or protein expression of involucrin and/or of ECM1, increases and/or maintains the barrier function of the skin and/or of the skin appendages and/or promoting cell differentiation in the skin and/or the mucous membranes and/or the skin appendages.

20. The cosmetic method according to claim 18, wherein the extract increases and/or maintains tissue renewal in the skin and/or the mucous membranes and/or the skin appendages.

21. The cosmetic method according to claim 18, wherein the method improves the surface appearance of the skin and/or the mucous membranes and/or the skin appendages.

22. The cosmetic method according to claim 18, wherein the method reduces hair loss and/or increases the radiance of the skin complexion.

23. The cosmetic method according to claim 18, wherein the method reduces water losses in the skin and/or the skin appendages and/or prevents the dehydration thereof.

24. The cosmetic method according to claim 18, wherein the extract of Cistus monspeliensis is an extract of the leaves.

25. The cosmetic method according to claim 18, wherein the extract of Cistus monspeliensis is an aqueous extract obtained in water or a mixture of water and a solvent chosen from the group consisting of an alcohol, a glycol, a polyol and a mixture thereof.

26. The cosmetic method according to claim 25, wherein the extract of Cistus monspeliensis is an aqueous extract obtained in water as the sole solvent.

27. The cosmetic method according to claim 18, wherein the cosmetic composition also comprises at least one cosmetically acceptable excipient, the extract being present in a concentration of from 0.0001% to 20% by weight, relative to the total weight of the cosmetic composition.

28. The cosmetic method according to claim 27, wherein the extract is present in the cosmetic composition in a concentration of from 0.001% to 5% by weight, relative to the total weight of the cosmetic composition.

29. The cosmetic method according to claim 18, wherein the administration is a topical application.

30. The cosmetic method according to claim 18, wherein the administration is a topical application to all or part of the face and/or the body and/or the scalp and/or the skin appendages and/or the mucous membranes, chosen from the legs, the hands, the thighs, the stomach, the neckline, the neck, the arms, the torso, the back, the hair and the face.

31. The cosmetic method according to claim 18, wherein the administration is a topical application to the neckline and/or the face.

32. The cosmetic method according to claim 18, wherein the administration is a topical application to the area around the eyes.

33. A treatment method for preventing and/or treating diseases induced by a decrease in the gene and/or protein expression of ECM1, and/or for preventing and/or treating scars comprising administering an effective amount of an extract of the aerial parts of Cistus monspeliensis or of a dermatological or pharmaceutical composition containing it to an individual.

34. The method according to claim 33, wherein the extract of Cistus monspeliensis is present in a dermatological or pharmaceutical composition in a concentration of from 0.0001% to 20% by weight, relative to the total weight of the composition.

35. The method according to claim 33, wherein the extract of Cistus monspeliensis is an extract of the leaves.

36. The method according to claim 33, wherein the diseases induced by a decrease in the gene and/or protein expression of ECM1 are lichen sclerosus or lipoid proteinosis.

37. The method according to claim 34, wherein the extract is present in the dermatological or pharmaceutical composition in a concentration of from 0.001% to 5% by weight, relative to the total weight of the composition.

Description

EXAMPLES

Example 1: Preparation of Extracts of C. Monspeliensis

[0112] The plant C. monspeliensis originates from Morocco.

[0113] Example 1a) 10% by weight of dried aerial parts of the plant C. monspeliensis, relative to the total weight of the plant parts and of the solvent, were macerated in water as sole solvent at a temperature of 80° C., for a period of 1 hour. The extract obtained was separated out by centrifugation and the supernatant was filtered (0.20 μm). The extract is in liquid form.

[0114] Example 16b) 20% by weight of dried aerial parts of the plant C. monspeliensis, relative to the total weight of the plant parts and of the solvent, were macerated in water as sole solvent at a temperature of 80° C., for a period of 1 hour. The extract obtained was separated out by centrifugation and the supernatant was filtered (0.20 μm). The extract is in liquid form.

[0115] Example 1c) 10% by weight of dried aerial parts of the plant C. monspeliensis, relative to the total weight of the plant parts and of the solvent, were macerated in a water/propanediol mixture (20:80; v/v) as sole solvent at a temperature of 4° C., for a period of 2 hours. The extract obtained was separated out by centrifugation and the supernatant was filtered (0.20 μm). The extract is in liquid form.

[0116] Example 1d) 10% by weight of dried leaves, relative to the total weight of the plant part and of the solvent, were macerated in water as sole solvent at a temperature of 4° C., for a period of 24 hours. The extract obtained was separated out by centrifugation and the supernatant was filtered (0.20 μm). The extract is in liquid form.

[0117] Example 1e) 10% by weight of dried aerial parts of the plant C. monspeliensis, relative to the total weight of the plant parts and of the solvent, were extracted into water under subcritical conditions, at a temperature of 120° C. under a pressure of 250 bar. The crude extract is decanted, centrifuged and then filtered. The extract is in liquid form.

[0118] Each of these extracts described above may then be dried and sprayed in the presence of maltodextrin (from 70% to 90% by weight, advantageously 80% by weight, relative to the total weight of maltodextrin and of the extract). The extract obtained after spraying will be in powder form.

Example 2: Increase in the Gene and Protein Expression of the Marker ECM1 in the Presence of an Extract of C. Monspeliensis

Example 2a) Increase in the Gene Expression of ECM1

[0119] Protocol. Human keratinocytes obtained from healthy female donors (from 39 to 60 years old) were cultivated until confluence was reached (5-10 days of culturing) in a defined growth medium (Dulbecco's Modified Eagle Medium: DMEM) supplemented with 10% foetal calf serum and antibiotics, in a controlled environment (37° C., 5% CO.sub.2, relative humidity>95%). The cells were then treated with the extract of C. monspeliensis prepared under the conditions described in Example 1a) at a final concentration in the medium of 0.001% by weight relative to the final volume of the medium, for a period of 48 hours, or were not treated with the extract (untreated control). The cells were recovered and frozen at −80° C. until the time of use.

[0120] The total RNA was extracted using the NucleoSpin 96 RNA kit (Machery-Nagel GmbH & Co KG). The quantification and quality of the total RNA were controlled by measuring the optical densities at 260 and 280 nm.

[0121] The expression of the ECM1 gene was quantified by qRT-PCR. The messenger mRNAs were transcribed into cDNA with specific oligonucleotides (SYBR Green) using a thermocycler (LightCycler®480 I Master system (Roche Molecular Diagnostics). The genes ACTB (Actin B) and EEF1A1 (Eukaryotic Translation Elongation Factor 1 Alpha 1) were used as reference genes for normalization of the results. The fluorescence was measured on each amplification cycle. The relative quantification was performed by means of the ΔΔCt method after producing the calibration curve.

[0122] The results are expressed as the mean±standard deviation of the mean on five different keratinocyte cultures and are indicated in Table 1 below. The statistical study of the differences was performed using Student's t test versus the untreated control (control (without extract)).

TABLE-US-00001 TABLE 1 MEAN SD Control (with out extract) 100  0 Extract of C. monspeliensis prepared according to Ex. 1a) 152* 9 (1 × 10.sup.−3% w/v) *P < 0.001

[0123] Conclusion: The extract of C. monspeliensis increased the gene expression of ECM1 by at least 43% relative to the control, showing its capacity for increasing cell differentiation, thus participating in tissue homeostasis.

Example 2b) Increase in the Protein Expression of ECM1

[0124] Protocol. Human keratinocytes obtained from a healthy female donor (from 39 to 60 years old) were cultivated in a defined medium (DMEM) until confluence was reached (4 days of culturing) and then placed or not placed in contact with the extract of C. monspeliensis prepared according to Example 1a) (final concentration of 1×10.sup.−3% by weight relative to the final volume of the medium) for a period of 48 hours. The same culture medium without the addition of extract was used as a control (untreated control).

[0125] The cells were subsequently harvested and then lysed with a specific lysis buffer in order to perform the immunolocalization (Western blotting). The total protein concentration was determined by means of the BCA method, and each condition is deposited at the same concentration. The proteins were identified by capillary electrophoresis (ProteinSimple, USA) using an anti-ECM1 primary antibody and immunolocalized using a peroxidase-coupled conjugated secondary antibody. The results were quantified using the Compass Software (version 2.7.1 (ProteinSimple)), and reported relative to the untreated control (control (without extract)). The results are expressed as the mean±standard deviation of the mean on two different keratinocyte cultures (n=4) and are indicated in Table 2 below. The statistical study was performed using the Sigmaplot™ software.

TABLE-US-00002 TABLE 2 MEAN SD Control (without extract) 100  2 Extract of C. monspeliensis prepared according to Ex. 1a) 139* 6 (1 × 10.sup.−3% w/v) *p < 0.001

[0126] Conclusion: The extract of C. monspeliensis increased the expression of ECM1 in the keratinocytes in culture by at least 31%, confirming the capacity of the extract for increasing cell differentiation and consequently tissue homeostasis.

Example 3: Increasing the Protein Synthesis of Involucrin in the Presence of an Extract of C. Monsneliensis

[0127] Protocol. Human keratinocytes obtained from healthy adult female donors (45 and 46 years old) were cultivated in a defined growth medium (MCDB153 standard medium) supplemented with 2% foetal calf serum in a controlled environment (37° C., 5% CO.sub.2, relative humidity>95%) for a period of 3 to 4 days. The culture medium was replaced with serum-free medium and treated or not treated with the extract of C. monspeliensis prepared according to Example 1a) (final concentration of 1×10.sup.−3% by weight relative to the final volume of the medium) for a period of 3 days.

[0128] The level of production of involucrin was quantified by means of the ELISA method on cell homogenates.

[0129] The results are expressed as the mean percentage±standard deviation relative to the untreated control (control (without extract)) (n=3) and are indicated in Table 3 io below. The statistical study was performed using the Sigmaplot™ software.

TABLE-US-00003 TABLE 3 MEAN SD Control (without extract) 100   15 Extract of C. monspeliensis prepared according to Ex. 1a) 390* 107 (1 × 10.sup.−3% w/v) *p < 0.01

[0130] Conclusion: The extract of C. monspeliensis increased the synthesis of involucrin by at least 168% in the cultivated keratinocytes. The extract of C. monspeliensis according to the invention is effective for increasing the barrier function of the skin and/or of the skin appendages.

Example 4: In Vivo Evaluation of Tissue Renewal in the Presence of an Extract of C. Monspeliensis

[0131] Protocol. The effect of a clinical treatment with a formulation comprising the extract of C. monspeliensis on tissue renewal was studied by using the DHA (dihydroxyacetone) method. The principle is to colour the skin surface (epidermis) by using this molecule used as a self-tanning molecule. The chemical reaction between DHA and the amino acids of the proteins of the epidermis forms brown-coloured melanoidin pigments. The colour was evaluated using a chromameter (Chromameter CR-400). The decolorization time was used to estimate the time required for the epidermis to become renewed.

[0132] Women from 45 to 65 years old of light phototype (Type I and II according to Fitzpatrick) were included in the analysis. The volunteers applied to each forearm, twice a day for a period of 28 days, a formulation comprising the extract of C. monspeliensis prepared under the conditions of Example 1a) (0.1% by weight relative to the total weight of the formulation of Example 5a) or of the same formulation not containing the extract according to the invention (control).

[0133] The parameter ΔE was calculated to measure the colour difference between a measurement at time x (Dx) and the measurement at time 0 (DO: basal reference level). The time required to reach a value ΔE=0 corresponds to the renewal time of the epidermis. The angle ITA was calculated to measure the difference in brown colour before and after application of the product.

[0134] The results correspond to the number of days required for total renewal of the epidermis (ΔE=0), after 28 days of application of the formulation comprising the extract of C. monspeliensis or without the extract (control), and are indicated in Table 4 below.

TABLE-US-00004 TABLE 4 MEAN Control 20.3  Extract of C. monspeliensis Example 1a) 0.1% (w/w) 16.9* *p < 0.1

[0135] Conclusion: After 28 days of application of the formulation comprising the extract of C. monspeliensis, the renewal time of the epidermis was reduced by 3.4 days in comparison with the control. The extract is thus effective for increasing tissue renewal.

Example 5: Examples of Cosmetic or Dermatological Compositions Comprising an Extract of Cistus Monspeliensis

Example 5a) Cosmetic Formulation

[0136] The percentages are expressed in Table 5 on a weight basis relative to the total weight of the cosmetic formulation.

TABLE-US-00005 TABLE 5 Water QS 100 Plant oil 4.00 Cocoyl caprylate/caprate 4.00 Dicaprylyl ether 4.00 Propylene glycol, phenoxyethanol, chlorphenesin, methylparaben 2.50 Sodium polyacrylate 1.00 Hydrogenated plant glycerides 1.00 Sodium stearoyl glutamate 0.05 Extract of C. monspeliensis Ex. 1a) 0.10

Example 5b) Anti-Ageing Cream

[0137] The percentages are expressed in Table 6 on a weight basis relative to the total weight of the cream (% w/w).

TABLE-US-00006 TABLE 6 Phase A Sucrose polystearate, cetyl palmitate 2.50 Sodium stearate glutamate 0.50 Cetearyl alcohol 1.00 Pentaerythrityl distearate 1.5 Dicaprylyl ether 3.00 Dibutyl adipate 5.00 Propylheptyl caprylate 5.00 Phenoxyethanol, ethylhexylglycerol 1.00 bis-Ethylhexyloxyphenol methoxyphenyl triazine 1.50 Phase B Water 64.40 Glycerol 3.00 Xanthan gum 0.30 Disodium EDTA 0.20 Phase C Methylenebis(benzotriazolyl)(tetramethylbutyl)phenol (nano), 6.00 water, decyl glucoside, propylene glycol, xanthan gum Phase D Undecane, tridecane 2.00 Fluorphlogopite, lauroyllysine 1.00 Phase E Water 2.00 Extract of C. monspeliensis Ex. 1a), maltodextrin 0.10

[0138] The cream is prepared by the usual methods in the field well known to those skilled in the art, by mixing the five phases.

Example 6: Increasing the Radiance and Homogeneity of the Complexion

[0139] Protocol. B16 melanocytes were cultivated in a standard culture medium comprising foetal calf serum for a period of 3 days at a temperature of 37° C. (5% CO.sub.2). The medium was renewed identically with addition of the extract of C. monspeliensis according to Example 1a) at a final concentration of 0.01% by weight relative to the final volume of the medium or without any extract (control medium). After a period of 3 days of culturing, the total amount of melanin was measured by optical density at 475 nm. The tests were performed in triplicate and the results expressed as the percentage mean relative to the control (culture without any extract of C. monspeliensis) (SD: standard deviation). The statistical analysis was performed by Student's test (Sigmaplot™)

TABLE-US-00007 TABLE 7 Results: MEAN SD Control 100 0 Extract of C. monspeliensis 0.01% w/v Ex. 1a)   38* 7 *p < 0.001

[0140] Conclusion: the extract reduced the total amount of melanin by at least 55% in the melanocytes analysed, demonstrating the effect of the extract on the homogeneity and radiance of the complexion.