Kapok tree flower extract, and cosmetic, pharmaceutical or dermatological compositions containing same

Abstract

The invention relates to an extract of flowers, preferably calyxes, from kapok trees, in particular Bombax costatum, to a process for preparing same and to the extract obtainable by said process. The invention also relates to a composition comprising such an extract, the composition being advantageously a cosmetic, pharmaceutical or dermatological composition. The invention also relates to such a composition or such an extract for use in preventing or treating disorders or diseases of the skin, the mucous membranes or keratinous appendages and for use in preventing or treating vascular disorders. The invention finally relates to a cosmetic care process for the skin, the keratinous appendages or the mucous membranes, with a view to improving the condition thereof or the appearance thereof, which process consists in administering such a composition or such an extract.

Claims

1. Cosmetic-method for treating dehydrated skin; skin with redness; elderly or photo-aged skin; photosensitive skin; cutaneous aging; and disorders related to radical attacks linked to chemical or atmospheric pollution, comprising administering to a subject in need thereof an effective amount of a polyphenol-rich extract of the Kapok tree flower comprising at least 15% by weight of polyphenols, relative to the total weight of the dry extract, at least 15% by weight of oligoproanthocyanidins (OPC), expressed as catechin equivalent, relative to the total weight of the dry extract, and at least 6% by weight of organic acids, relative to the total weight of the dry extract, wherein the Kapok tree is Bombax costatum.

2. The method of claim 1, wherein the extract further comprises at least 5% by weight of flavonoids, expressed as rutin equivalent, relative to the total weight of the dry extract.

3. The method of claim 1, wherein the extract is an extract of Kapok tree calyxes.

4. The method of claim 1, wherein the organic acids are alpha-hydroxy acids.

5. The method of claim 1, wherein the organic acids are acetic acid, malic acid, lactic acid, citric acid or mixtures thereof.

6. The method of claim 1, wherein the extract is obtained by solid/liquid extraction of the flowers of the Kapok tree in a solvent chosen from amongst binary mixtures of water/glycol.

7. The method of claim 6, wherein the solvent is chosen from amongst binary mixtures of water/glycol in a proportion comprised between 30 and 90% of glycol in water.

8. The method of claim 6, wherein the glycol is propanediol or propylene glycol.

9. The method of claim 1, wherein the cutaneous aging is photo aging.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1 represents the contractile forces developed by wrinkle fibroblasts (WF) compared to healthy fibroblasts (HF).

(2) FIG. 2 represents the contractile forces developed by healthy fibroblasts (HF) in the presence or not of the KPO extract according to the invention.

(3) FIG. 3 represents the contractile forces developed by wrinkle fibroblasts (WF) in the presence or not of the KPO extract according to the invention.

(4) The following examples help to illustrate the invention.

EXAMPLE 1: EXTRACTS ACCORDING TO THE INVENTION

(5) Polyphenol-rich extracts of Bombax costatum calyxes are obtained according to the following method: a) ground Bombax costatum calyxes are put in solution in a mixture of water/propanediol 30/70, 20/80 or 10/90 w/w b) extraction 2 h at 50° C. c) solid/liquid separation by successive filtrations.

(6) The polyphenol-rich liquid extract obtained in this way has the following characteristics (% by weight of molecules/dry extract of the extract obtained):

(7) TABLE-US-00001 Mixture of water/propanediol (w/w) 30/70 20/80 10/90 Dry extract(2 h, 105° C., ventilated oven) 1.6%  1.2% 1.3%  Total polyphenols (eq. Gallic acid)  19% .sup. 23% 22% OPC (eq. Catechin) 20.6%  22.5% 24% Flavonoids (eq. Rutin) 4.6%   6%  5% Antioxidant activity (DPPH) 31 (μg) 12 (μg) 27(μg)

(8) The Kapok tree extract according to the invention obtained according to example 1 (20/80) is also referred to as KPO extract in the text and Figures.

EXAMPLE 2: EXTRACT ACCORDING TO THE INVENTION

(9) A polyphenol-rich extract of Bombax costatum calyxes is obtained according to the following method: a) ground Bombax costatum calyxes are put in solution in a mixture of water/ethanol 20/70 w/w; b) extraction 2 h at 75° C.; and c) solid/liquid separation by successive filtrations.

(10) The polyphenol-rich liquid extract obtained in this way has the following characteristics (% molecules/dry extract of the extract obtained):

(11) TABLE-US-00002 Dry extract (2 h, 105° C., ventilated oven) 1.05% Total polyphenols (eq. Gallic acid)   24% OPC (eq. Catechin)   14% Flavonoids (eq. Rutin)  7.8% Antioxidant activity (DPPH) 34 (μg)

EXAMPLE 3: BIOLOGICAL ACTIVITY TESTS OF THE EXTRACT ACCORDING TO THE INVENTION

(12) In the example below, the extract tested, called KPO extract, is an extract according to the invention obtained according to example 1 (mixture of water/propanediol 20/80).

(13) 1—Screening of Gene Expression in Fibroblast, Keratinocyte and Skin Explant Cultures

(14) The effect of KPO extract on messenger RNA expression characteristic of cosmetic requirements was evaluated in characteristic skin models: normal human epidermal keratinocytes or normal human dermal fibroblasts were incubated for 24 hours with 0.0005% and 0.001% KPO extract (dry extract concentration: d.e.). Additionally, skin explants were incubated for 24 h with 0.005% and 0.001% KPO extract (d.e.). At the end of incubation, the mRNA was extracted and the expression of 90 genes of interest was analysed by RT-qPCR.

(15) The results showed potential biological activity of the KPO extract in the following areas: Neosynthesis, structuring and remodelling of the dermal matrix: stimulation of DCN, SPARC, BGN, COL3A1, TP63, TGFB1 genes; Adhesion and barrier function: stimulation of CD44, CTNNA1, ITGA2, SDC1, LAMC2, TNC, COL17A1, VCAN, PXN, LAMAS, S100A7 genes; Longevity and renewal: stimulation of SIRT6, ATP5A1, INSR, EGF, SIRT3 genes; Pigmentation: stimulation of POMC and PMEL genes; Detoxification and antioxidant defence: stimulation of BSG, CAT, GLRX, MSRA, MSRB2, TXN, SOD2, PPIA, HMOX1, HSPB1, DGAT1, GBA genes.

(16) To summarize, two main effects of the KPO extract emerge from this analysis. The first is adhesion and the barrier function with strong expression of the genes involved in cell-cell or cell-matrix adhesion. This activity is boosted by extracellular matrix neosynthesis and structuring activities which show an effect on tissue firmness. The stress response is also very much present with the induction of many genes some of which show in particular strong induction. In this stress response, protection against oxidative stress is predominant. Reaction pathways to other types of stress and detoxification pathways are also activated.

(17) 2—Gene Expression of Markers of the Dermal Extracellular Matrix and of Cell Communication

(18) The effect of the KPO extract was evaluated on the gene expression of markers involved in cell communication and in the organisation of the dermal extracellular matrix in the fibroblasts: normal human dermal fibroblasts were incubated for 6 hours or 24 hours with 0.0005% or 0.001% KPO extract (d.e.) or TGFβ1 at 5 ng/ml (positive control for dermal matrix induction markers). At the end of incubation, the gene expression of the following markers was analysed by RT-qPCR. CTNNA1 (Catenin α): binds to cadherins to promote cellular interactions. Syndecan: proteoglycan which consolidates cell/cell and cell/matrix interactions. Laminin 5 (α3β3γ2): a molecule that assists adhesion to the basal membrane. TNC (Tenacin C): extracellular matrix proteins involved in cohesion of the dermo-epidermal junction and which promote the synthesis of collagen VII. SPARC: protein associated with various constituents of the extracellular matrix and which contributes to its organisation. BGN (Biglycan): proteoglycan involved in the assembly of collagen fibres. Versican: a major component of the ECM which plays a central role in the morphogenesis and maintenance of tissues. It also contributes to the formation of the elastic fibres of the skin. The collagens which give the dermis its firmness and resistance to pressure qualities, representing around 70% of the dermal Extra Cellular Matrix (ECM) components, the majority being of type I collagen (85-90% of dermal collagen) which is frequently associated with type III collagen. Type IV and VII collagen: glycoproteins, constituents of the Dermo-Epidermal Junction. Fibronectin: is a high-molecular weight glycoprotein. It plays a role in the structure of the dermal ECM. The elastic fibres are responsible for skin elasticity. They result from the assembly of two major components: elastin (1 to 3% of the dermis) and the microfibrils. LOXL: responsible for the assembly of elastin fibres. Decorin: proteoglycan involved in the assembly of the matrix. Dermatopontin: plays a role in the assembly of collagen fibres; it is important for maintaining the structural arrangement of the dermal extracellular matrix and tissue flexibility. Laminin 5 (α3β3): a molecule that assists adhesion to the basal membrane (Dermo-Epidermal Junction).

(19) The results are given as relative quantities (RQ) with respect to the untreated control. The significance of the results was evaluated using a statistical test (one-factor variance analysis followed by a Dunnet test): *p<0.05; **p<0.01; ***p<0.001; ns: non-significant p>0.05.

(20) The results are grouped together in tables 1 and 2 below. They show a considerable inductive effect of the KPO extract on the set of markers studied, suggesting a strong efficacy potential in structuration of the dermal matrix, Dermo-Epidermal Junction (DEJ) and the cell communication method. The KPO extract thus plays a role in hydration, elasticity, dermal exchanges (EDJ) and skin aging.

(21) TABLE-US-00003 TABLE 1 Analysis of gene expression after 6 h of treatment: Degree of expression (Relative Quantity) Positive Control Negative (TGFβ1 Gene evaluated Control 5 ng/ml) KPO 0.0005% KPO 0.001% SPARC 1.00 1.28 ns  .sup. 1.37 *** 1.60 **  BGN 1.00 1.28 ns .sup. 1.73 ** 2.11 *** CTNNA1 1.00 1.07 ns 1.48 *.sup.  1.48 *** Syndecan 1.00 1.85 ns 1.41 ns 1.73 ns .sup.  TNC 1.00 0.94 ns 1.41 ns 1.73 *  Versican 1.00 1.61 ns 1.15 ns 1.24 ns .sup.  Collagen I 1.00 0.84 ns 1.33 ns 2.08 *** Collagen III 1.00 0.91 ns 1.24 *.sup.  1.96 *** Collagen IV 1.00  1.68 *** 1.33 ns 1.55 **  Collagen VII 1.00  1.96 *** 0.86 ns 1.41 *  Fibronectin 1.00 1.09 ns 1.22 ns 1.60 **  Elastin 1.00 0.72 ns 1.26 ns 1.96 *** LOXL 1.00 1.05 ns .sup. 1.46 ** 1.99 **  Decorin 1.00 n.d. 1.29 *.sup.  1.30 *  Dermatopontin 1.00 1.14 ns 1.32 *.sup.  1.49 *  Laminin 5α3 1.00 n.d. 2.17 *.sup.  1.64 ns .sup.  Laminin 5β3 1.00 n.d. 1.48 *.sup.  1.61 * 

(22) TABLE-US-00004 TABLE 2 Analysis of gene expression after 24 h of treatment: Degree of expression (Relative Quantity) Positive Control Negative (TGFβ1 Gene evaluated Control 5 ng/ml) KPO 0.0005% KPO 0.001% SPARC 1.00  2.00 ***  1.65 *** 2.12***  BGN 1.00  2.92 ***  2.05 *** 2.61 **  CTNNA1 1.00 1.07 ns 1.47 * 1.80 **  Syndecan 1.00  8.08 *** 1.98 * 2.65 *  TNC 1.00 1.79 *  .sup. 1.53 ns 1.72 *  Versican 1.00 2.87 **  1.45 ** 1.06 ns .sup.  Collagen I 1.00 2.30 *  2.31 * 4.91 *** Collagen III 1.00 1.24 ns .sup. 1.35 ns 3.16 *** Collagen IV 1.00 2.81 **  1.60 ** 2.08 *** Collagen VII 1.00  3.07 *** .sup. 1.20 ns 1.78 ns .sup.  Fibronectin 1.00 2.12 *  .sup. 1.27 ns 1.77 *** Elastin 1.00 3.65 ** 2.50 * 6.20 *** LOXL 1.00 1.10 ns 1.39 * 2.28 *** Decorin 1.00 n.d.  1.19 *** 1.58 *  Dermatopontin 1.00 1.36 *  1.41 * 2.00***  Laminin 5α3 1.00 n.d. 1.58 * 1.60 *  Laminin 5β3 1.00 n.d. .sup. 1.35 ns 1.68 * 

(23) 3—Evaluation of Anti-Protease Activity in Dermal Fibroblasts

(24) The KPO extract was evaluated for its capacity to limit the production of MMP1 (Matrix MetalloProteinase-1) triggered by inflammatory stress in the dermal fibroblasts. Normal human dermal fibroblasts were pre-incubated for 24 hours with KPO at 0.0005% or 0.001% (d.e.). The fibroblasts were then stimulated by 100 nM of PMA (Phorbol Myristate Acetate) and incubated overnight with KPO extract. The production of MMP1 was evaluated by ELISA assay in cell supernatants. The results are given in pg of MMP1 per mg of total proteins (determined by BC Assay). The significance was evaluated by means of one-factor variance analysis followed by a Tukey test: *p<0.05; ***p<0.001; ns: non-significant p>0.05.

(25) The results are grouped together in table 3 below. The KPO extract significantly inhibited MMP1 release triggered by PMA stress in the fibroblasts; this result shows a protective effect of the dermal matrix and this a chronological and actinic anti-aging effect.

(26) TABLE-US-00005 TABLE 3 Production of MMP1 in fibroblasts stimulated by PMA: MMP1 (pg/mg of proteins) Control 3.859 ± 0.623 PMA 11.358 ± 2.560  +194% vs control *** KPO at 0.0005% 8.230 ± 0.768 −28% vs PMA (ns) KPO at 0.001% 7.727 ± 0.770 −32% vs PMA *

(27) 4—Evaluation of Anti-Inflammatory Potential

(28) The anti-inflammatory activity of the KPO extract was evaluated in keratinocytes stimulated by PMA treatment.

(29) Normal human epidermal keratinocytes were pre-incubated for 24 hours with KPO extract at 0.001%, 0.002% or 0.005% (d.e.) or reference anti-inflammatory molecules dexamethasone at 0.1 μM or indomethacin at 0.1 μM. The keratinocytes were then stimulated by 10 μg/ml of PMA (Phorbol Myristate Acetate) and incubated overnight with KPO extract or reference molecules. The production of inflammatory mediators Interleukin-8 (IL*), Interleukin-1β (IL1β) and Prostaglandin E2 (PGE2) was evaluated by ELISA assay in cell supernatants. The significance was evaluated by means of one-factor variance analysis followed by a Tukey test: *p<0.05; ***p<0.001; ns: non-significant p>0.05.

(30) The results are grouped together in tables 4, 5 and 6 below. The KPO extract significantly inhibited the release of cytokines IL1β and IL8, and to a lesser degree PGE2, triggered by pro-inflammatory stress in the keratinocytes. These results show the anti-inflammatory potential of the KPO extract and an anti-aging effect on sensitive skin, or even on irritated skin.

(31) TABLE-US-00006 TABLE 4 Production of IL8 in keratinocytes stimulated by PMA: IL8 (pg/ml)/OD neutral red Standard Mean deviation Control 47.215 4.711 PMA 1420.626 92.557 +2909% vs Control *** KPO at 0.001% 1198.008 154.124 −16% vs PMA * KPO at 0.002% 795.287 84.255 −44% vs PMA *** KPO at 0.005% 338.854 18.731 −76% vs PMA ***

(32) TABLE-US-00007 TABLE 5 Production of IL1β in keratinocytes stimulated by PMA: IL1Beta (pg/ml)/OD neutral red Standard Mean deviation Control 0.007 0.000 PMA 0.040 0.006 +451% vs Control *** Dexa + PMA 0.026 0.002 −35% vs PMA ** KPO at 0.001% 0.018 0.003 −55% vs PMA *** KPO at 0.002% 0.017 0.003 −57% vs PMA *** KPO at 0.005% 0.017 0.003 −57% vs PMA ***

(33) TABLE-US-00008 TABLE 6 Production of PGE2 in keratinocytes stimulated PMA: PGE2 (pg/ml)/OD neutral red Standard Mean deviation Control 220.878 18.449 PMA 802.843 75.214 +263% vs Control *** Dexa + PMA 617.645 61.477 −23% vs PMA ** Indo + PMA 181.780 15.506 −77% vs PMA *** KPO at 0.001% 631.360 24.516 −21% vs PMA * KPO at 0.002% 701.909 60.353 −13% vs PMA (ns) KPO at 0.005% 735.274 60.135 −8% vs PMA (ns)

(34) 5—Evaluation of Antioxidant Potential

(35) The antioxidant potential of the KPO extract was evaluated in an assay model of intracellular ROS (Reactive Oxygen Species) induced by pro-oxidising H.sub.2O.sub.2 stress. Normal human epidermal keratinocytes were pre-incubated for 24 hours with KPO extract at 0.001%, 0.002% or 0.005% (d.e.) or reference anti-inflammatory molecules Quercetin at 10 μM or Vitamin C at 500 μM. The cell layer was then contacted with a DCFH-DA probe prior to induction of oxidative stress by 100 μM of H.sub.2O.sub.2. The amount of ROS produced was evaluated by measuring the fluorescence emitted when the probe is oxidised. The results were analysed statistically by one-factor variance analysis followed by a Tukey test: **p<0.01; ***p<0.001.

(36) The results are grouped together in table 7 below. The KPO extract significantly inhibited the production of ROS triggered by the presence of H.sub.2O.sub.2. These results demonstrate a powerful antioxidant potential. The KPO extract can therefore be advantageously used against pollution and aging.

(37) TABLE-US-00009 TABLE 7 Production of ROS in keratinocytes subjected to H.sub.2O.sub.2 stress: ROS (Arbitrary Unit) Standard Mean deviation Control 18564.862 12917.522 H.sub.20.sub.2 at 100 μM 133342.523 52149.942 +618% vs control *** Quercetin at 10 μmol/l 12959.361 2587.250 −90% vs H.sub.2O.sub.2 *** Vit C 500 μM 12807.703 4220.635 −90% vs H.sub.2O.sub.2 *** KPO at 0.001% 68750.713 10417.179 −48% vs H.sub.2O.sub.2 ** KPO at 0.002% 59761.384 15339.601 −55% vs H.sub.2O.sub.2 *** KPO at 0.005% 26779.475 6550.110 −80% vs H.sub.2O.sub.2 ***

(38) 6—In Vitro Evaluation of an Anti-Wrinkle and Firming Effect

(39) The «firming» effect of KPO extract was evaluated in a GlasBox® system on fibroblasts from the base of a wrinkle and on fibroblasts from adjacent non-wrinkled skin («healthy» fibroblasts). The GlasBox® system makes it possible to measure, within a dermal equivalent, the contractile force developed by the fibroblasts. Fibroblasts originating from the base of a wrinkle have less contraction force than those from adjacent non-wrinkled skin and in this way it is possible to evaluate the «anti-wrinkle» effect of a compound.

(40) Dermal equivalents were prepared by mixing fibroblasts (healthy skin or wrinkled skin) with a collagen solution. The fibroblasts originated from a sample obtained from the same patient (65-year-old woman) from the depth of the wrinkle, on the one hand, and from a nearby non-wrinkled skin. This mixture was cooled in rectangular GlasBox® tanks. A medium containing or not containing KPO extract at 0.005% or 0.001% (d.e.) was added.

(41) Two flexible silicon blades were introduced into each of these tanks. The dermal equivalent therefore develops between two blades equipped with a strain gauge («sensor»). Under the effect of the retraction force developed by the fibroblasts, the silicon blades are deformed; this is seen by a variation in the electrical resistance value of the strain gauge. This variation, measured in real time, is representative of the force developed within the dermal equivalent. The isometric forces were thus measured for 24 hours.

(42) The results are grouped together in FIGS. 1 to 3 and table 8. As expected, the contractile forces developed by the wrinkle fibroblasts (WF) are significantly reduced relative to those developed by healthy fibroblasts (HF).

(43) In wrinkle fibroblasts (WF), the KPO extract at 0.0005% increased the contractile forces significantly. This result demonstrates an anti-wrinkle effect.

(44) Applied to healthy fibroblasts, the KPO extract significantly increased the contractile forces. Analysis of the Glasbox® graphs shows that the AUC (area under the curve) and maximum contraction increased significantly in the presence of the extract. These results demonstrate a firming effect of the extract.

(45) TABLE-US-00010 TABLE 8 Analysis of GlasBox graphs (HF) Parameters Control HF HF + 0.001% KPO calculated Mean sem Mean sem AUC 2278.6 40.2 2574.5** 101.1 Max 97.79 1.46 112.44** 4.32 **p < 0.01 vs Control HF—One-factor Variance Analysis followed by a Fisher test

(46) 7—Evaluation of the Effect of KPO Extract on Mitochondrial Activity in Skin Fibroblasts

(47) Normal human dermal fibroblasts were incubated for 24 hours with KPO extract at 0.001% (d.e.). At the end of the treatment, the cells were collected by trypsination and incubated in the presence of an oxygen-sensitive fluorescent probe in order to measure oxygen consumption in real time by spectrofluorometry.

(48) The results are grouped together in table 9. These results show that the KPO extract triggers and increase in overall respiration. Thus, the KPO extract triggers stimulation of the respiratory chain activity, an effect which is representative of stimulation of mitochondrial functions.

(49) TABLE-US-00011 TABLE 9 Measurement of oxygen consumption in NHDF O2 consumption (%) Untreated control 100 KPO at 0.001% 145.40