Polysaccharide-Rich Bombax Costatum Flower Extract

Abstract

The invention relates to an extract from the flowers, preferably from the calyxes, of Bombax costatum, to a process for preparing same and to the extract obtained by said process. The invention also relates to a composition comprising such an extract, the composition advantageously being a cosmetic, pharmaceutical or dermatological composition. The invention also relates to such a composition or such an extract for the use thereof in preventing or treating disorders or pathological conditions of the skin, the mucous membranes or the hair and nails, as well as the imbalance or disorders associated with the imbalance of the microbiota of the skin, the mucous membranes, the hair, the nails and associated skin appendages. Finally, the invention relates to a cosmetic care method for the skin, the hair and nails or the mucous membranes, with a view to improving the condition or the appearance thereof, which method consists in administering such a composition or such an extract.

Claims

1.-10. (canceled)

11. A polysaccharide-rich extract from flowers of Bombax costatum, comprising at least 15% of polysaccharides relative to the total weight of the dry extract, the polysaccharides having an apparent molecular mass comprised between 1500 and 6000 kDaltons.

12. The extract according to claim 11, comprising at least 30% by weight of polysaccharides, relative to the total weight of the dry extract.

13. The extract according to claim 11, wherein the polysaccharides comprise monosaccharides and derivatives selected from the group consisting of galactose, rhamnose, galacturonic acid, glucuronic acid, and mixtures thereof.

14. The extract according to claim 11, wherein the polysaccharides comprise 10 to 16% galactose; 10 to 17% rhamnose; 10 to 17% galacturonic acid; and 3 to 7% glucuronic acid.

15. The extract according to claim 11, wherein the extract is obtained by solid/liquid extraction of the flowers of Bombax costatum, in water.

16. A process for preparing a polysaccharide-rich extract from flowers of Bombax costatum comprising at least 15% of polysaccharides relative to the total weight of the dry extract, the polysaccharides having an apparent molecular mass comprised between 1500 and 6000 kDaltons, wherein the process comprises at least one solid/liquid extraction step in water.

17. The process according to claim 16, further comprising the following successive steps: a) crushing the flowers of Bombax costatum, b) extracting the flowers crushed in step a) in water; c) separating the solid phase and the liquid phase obtained in step b) by decantation, and/or centrifugation and/or precipitation and/or successive filtrations.

18. A composition comprising, as active ingredient, a polysaccharide-rich extract from flowers of Bombax costatum, comprising at least 15% of polysaccharides relative to the total weight of the dry extract, the polysaccharides having an apparent molecular mass comprised between 1500 and 6000 kDaltons.

19. A method for preventing and/or treating: imbalances and/or disorders related to the imbalance of the microbiota of the skin and/or mucous membranes and/or skin appendages, and/or disorders or pathological conditions of the skin and/or mucous membranes and/or skin appendages, comprising administering to a subject in need thereof an effective amount of a polysaccharide-rich extract from the flowers of Bombax costatum, comprising at least 15% of polysaccharides relative to the total weight of the dry extract, the polysaccharides having an apparent molecular mass comprised between 1500 and 6000 kDaltons.

20. A cosmetic care method for improving the condition and/or the appearance of the skin and/or skin appendages and/or mucous membranes on healthy subjects, comprising administering a polysaccharide-rich extract from the flowers of Bombax costatum, comprising at least 15% of polysaccharides relative to the total weight of the dry extract, the polysaccharides having an apparent molecular mass comprised between 1500 and 6000 kDaltons.

21. The extract according to claim 11, wherein the polysaccharides comprise a mixture of galactose, rhamnose, galacturonic acid, and glucuronic acid.

22. The process according to claim 16, further comprising the following successive steps: d) bleaching the liquid phase obtained in step c) using a suitable adjuvant; e) drying the liquid phase obtained in step c) or d); and f) physically and microbiologically stabilizing the extract obtained in step c), d) or e).

23. The method according to claim 19, wherein: the imbalances and/or disorders related to the imbalance of the microbiota of the skin and/or mucous membranes and/or skin appendages are selected from atopic dermatitis, eczema, the development of bad underarm odors, the weakening of the skin barrier, acne, psoriasis, hidradenitis suppurativa, folliculitis, cradle cap, dandruff, itching, irritations, candidiasis and bacterial vaginosis, and the disorders or pathological conditions of the skin and/or mucous membranes and/or skin appendages are selected from inflammatory reactions, oxidation reactions, disorders related to radical attacks related or not to pollution and/or related to exposure to UV or IR, photosensitized skin, disorders or pathological conditions related to microbial attacks, barrier or homeostasis disorders, aging, disorders or pathological conditions related to mechanical and/or thermal aggressions on the skin and/or mucous membranes and/or skin appendages.

24. The cosmetic care method according to claim 20, for preventing and/or treating alterations to the skin barrier; dehydrated skin; the skin with redness; aged or photo-aged skin; skin aging, in particular photo-aging; disorders related to mechanical or thermal aggressions on the skin and disorders related to radical attacks related to chemical or atmospheric pollution.

Description

DESCRIPTION OF THE FIGURES

[0112] FIG. 1 represents the growth curves of C. acnes and M. furfur (see Example 2-V-b.).

[0113] FIG. 2 represents the effect of the BCP active ingredient on the bacterial growth of different strains in co-culture as a function of time (see Example 2-V-b.).

[0114] FIG. 3 represents the analysis of the morphology of the RHEs after Hematoxylin/Eosin staining (see Example 2-VI-b.).

[0115] FIG. 4a and FIG. 4b represent the growth curves of the lactobacillus strains (see Example 2-VII-b.).

[0116] FIG. 5 represents the analysis of the formation of biofilm by different strains of the microbiota of the skin in the presence of the BCP active ingredient (see Example 2-IX).

EXAMPLES

Example 1: Preparation of a Solution of Polysaccharides from Calyxes of Bombax costatum

[0117] The Bombax costatum Calyxes are ground, then suspended with stirring in water at a proportion of 2% w/w calyxes/water; [0118] Extraction for one hour with stirring at 90° C.; [0119] Centrifugation of the solution obtained in order to separate the solid residues of the plant; [0120] Bleaching by addition of activated carbon and filtration; [0121] Concentrate of polysaccharides by Ultrafiltration 15 kDa; [0122] Addition of glycerin and evaporation of the water under vacuum in order to obtain a final glycerin concentration of 80% w/w.

[0123] Analytical profile of the polysaccharide solution obtained [0124] Orange viscous solution [0125] Dry matter (m/m): 1.24% [0126] pH: 6.3 [0127] Ash 8.9% [0128] Total sugars (determination by the anthrone method): 35% [0129] Viscosity of the solution (TA, mobile1, 2 rpm): 2592 Cps

[0130] Analytical profile of the polysaccharides obtained (dry matter): [0131] Average molecular mass (determination by Gas Phase Chromatography): 3711 kDa [0132] Composition (determination by Gas Phase Chromatography): 15% Galactose; 17% rhamnose; 17% galacturonic acid; 5% glucuronic acid

Example 2: Biological Activities

[0133] The potential biological activities of the extract were investigated by a gene expression modulation test on dermal fibroblasts and melanized reconstructed epidermis. Thus, the expression of 96 genes of major interest in skin and cosmetic physiology was studied by PCR-array on fibroblasts and melanized reconstructed epidermis.

[0134] a. Materials and Methods:

[0135] The Bombax costatum polysaccharide extract according to Example 1 (called BCP extract) at 0.05% dry matter was added to the culture medium of normal human dermal fibroblasts (NHDFs) or of reconstituted melanized human epidermis.

[0136] After 6 hours or 24 hours of incubation, the expression of the selected markers was evaluated by quantitative RT-PCR (TaqMan microfluidic card). The variation in expression of the markers studied compared to the control was expressed in relative quantity (RQ, RQ>1: increase, RQ<1: decrease).

[0137] b. Results:

[0138] The most significant results showing the effect of BCP extract on gene expression in reconstructed epidermis are shown in Table 1 below.

TABLE-US-00001 TABLE 1 Variations in the expression of genes of interest in melanized reconstructed human epidermis BCP 0.05% dm RQ p value RAB11A Ras-related protein Rab-11A 1.7372 0.0073 SMPD1 Sphingomyelin phosphodiesterase 1.4885 0.0012 (Acid sphingomyelinase) KRT19 Keratin, type I cytoskeletal 19 1.3016 0.0321 (keratin 19) SDC1 Syndecan-1 1.279 0.0401 MITF Microphthalmia-associated 0.7409 0.0298 transcription factor Relative Quantity (RQ) compared to the control = 1 p value determined following a Student test

[0139] These results show that the BCP extract, by varying the gene expression of some markers, has a particular interest in the following activities:

[0140] Synthesis and Remodeling of Lipids in the Barrier Function of the Epidermis

[0141] The BCP extract increases the gene expression of 2 enzymes involved in the synthesis or remodeling of lipids within the stratum corneum: The RAB11A gene for a GTPase (Ras-related protein Rab-11A) and the SMPD1 gene for sphingomyelin phosphodiesterase. By increasing the expression of these two genes, the BCP active ingredient allows to reinforce the barrier function of the epidermis and its hydration.

[0142] Mechanisms Regulating Cell Adhesion, Migration, Proliferation and Differentiation of Keratinocytes:

[0143] The BCP active ingredient increases the expression of syndecan-1 (SDC1).

[0144] Stem Cell Protection:

[0145] Keratin 19 encoded by the KRT19 gene is an epithelial marker considered as a marker of epidermal stem cells.

[0146] By stimulating the expression of KRT19, the BCP extract has a protective effect on stem cells.

[0147] Inhibition of Melanogenesis:

[0148] The BCP extract causes a decrease in the expression of the MITF gene. Thus, by decreasing the expression of the MITF gene, the BCP extract has melanogenesis inhibitory activity.

[0149] Table 2 below shows the most significant results of the BCP extract on gene expression in fibroblasts.

TABLE-US-00002 TABLE 2 Variations in the expression of genes of interest in normal human dermal fibroblasts (NHDFs) BCP 0.05% dm RQ p value SOD2 Superoxide dismutase 2, 9.4811 0.0045 mitochondrial MT1G Metallothionein-1G 5.1496 0.0037 FBN2 Fibrillin-2 2.9182 0.026 FOXO1 Forkhead box protein O1 2.0353 0.0118 CSGALNACT1 Chondroitin sulfate N- 2.0195 0.0055 acetylgalactosaminyltransferase 1 MKI 67 Antigen Ki-67 1.9088 0.0062 DCN Decorin (PGS2) 1.9083 0.0332 CTGF Connective tissue growth factor 1.8602 0.0377 TXNRD1 Thioredoxin reductase 1 1.664 0.0148 ELN Elastin (tropoelastin) 1.6088 0.0226 LMNB1 Lamin-B1 1.5846 0.0068 FGF2 Heparin-binding growth factor 2 1.5844 0.0355 (Fibroblast growth factor 2) FBN1 Fibrillin-1 (Marfan syndrome) 1.4919 0.0119 COL3A1 Collagen 3 alpha 1 subunit 1.4364 0.0362 (COL3A1) CAT Catalase 1.2865 0.0387 ACTA2 Actin, aortic smooth muscle 1.2722 0.0293 CHST15 Carbohydrate sulfotransferase 15 1.2604 0.0194 FBLN5 Fibulin-5 1.2554 0.0391 COL16A1 Collagen 16 alpha 1 subunit 1.2117 0.0422 (COL16A1) FTH1 Ferritin heavy chain 1.205 0.0242 PXN Paxillin 1.1758 0.0184 TLN Talin-1 1.1482 0.0275 SESN2 Sestrin-2 0.5513 0.0406 MMP1 Matrix metalloproteinase 1 0.5478 0.0045 (interstitial collagenase) PTGS2 Prostaglandin G/H synthase 2 0.3023 0.0314 Relative Quantity (RQ) compared to the control = 1 p value determined following a Student test

[0150] These results show an activity of the BCP extract in the following areas:

[0151] Protection Against Oxidative Stress:

[0152] The BCP extract induces the expression of several enzymes involved in antioxidant defense: superoxide dismutases, in particular superoxide dismutase 1 (Cu/ZnSOD) encoded by the SOD1 gene and superoxide dismutase 2 (MnSOD) encoded by the SOD2 gene.

[0153] The BCP active ingredient also induces the expression of one of the 2 isoforms of metallothionein 1 (MT1G).

[0154] The BCP active ingredient also induces the TXNRD1 gene encoding for the isoform 1 of thioredoxin reductase.

[0155] The joint induction of these genes coding for proteins with antioxidant and/or detoxifying activity confers protection against UVs and heavy metals from, for example, urban pollution.

[0156] The Extracellular Matrix and the Elasticity of the Skin for an Anti-Aging Effect:

[0157] The main function of fibroblasts present in the dermis is to produce, degrade, and therefore regulate the components of the extracellular matrix (ECM) with which they interact. The ECM is a complex structure formed by a network of collagen fibers, elastin fibers and structural glycoproteins.

[0158] Among the ECM proteins regulated by the BCP active ingredient, it is possible to find elastin (ELN) but also fibrillins-1 and -2 encoded respectively by the FBN1 and FBN2 genes. In addition to the induction of ELN and FBN1 genes, the BCP active ingredient also induces FBN2, also constituting the microfibrils.

[0159] In parallel with its action on the elastic fibers, the BCP active ingredient also acts on the collagen fibers since it increases the expression of the alpha 1 subunit of collagen 3 (COL3A1).

[0160] The joint action of BCP on the expression of the constituents of elastin and collagen fibers as well as on MMP1 and paxillin goes in the direction of an effect on the elasticity of the skin, which is particularly interesting in an anti-aging context.

[0161] Moreover, it has been demonstrated that skin aging is associated with a decrease in the proliferation of fibroblasts. There is a decrease in expression of the proliferation factor Ki-67 related to age (Ma, C. and al., 2011. Expression of metallothionein-I and II in skin ageing and its association with skin proliferation. Br. J Dermatol., 164(3), pp. 479-482). The BCP active ingredient increases the expression of MKI67, indicating an increase in cell proliferation, which reinforces its anti-aging effect.

[0162] The Nuclear Lamin:

[0163] It was shown that the expression of lamin B1 (LMNB1) in the skin decreases with age. Lamin B1 is thus considered to be a marker of skin cell senescence. Indeed, it has been shown that senescence related to age or associated with skin pathological conditions is accompanied by a loss of expression, at the protein and mRNA level, of lamin B1 (Dreesen, O. and al., 2013. The contrasting roles of lamin B1 in cellular aging and human disease. Nucleus, 4(4), pp. 283-290).

[0164] Consequently, an increase in the expression of LMNB1 within dermal fibroblasts indicates an effect of the BCP active ingredient reducing senescence which may be associated with age or skin pathological conditions, and on the other hand proves to be beneficial for maintaining core structure and integrity.

[0165] Autophagy:

[0166] The BCP extract induces a decrease in the expression of SESN2. SESN2 in particular is involved in the UV response of skin cells. This goes in the direction of a greater activity of the mTORC1 complex which goes hand in hand with a decrease in the autophagic activity of the cells and thus a regulatory effect of the mitophagic activity.

[0167] Skin Healing:

[0168] Within focal adhesion sites, integrins are bonded, via adhesion proteins, to intracellular actin filaments. Among these cytoplasmic adhesion proteins, it is in particular possible to find talin-1 encoded by the TLN1 gene overexpressed by the BCP active ingredient. It constitutes the initial bond between integrins and the actin cytoskeleton. The binding of talin to integrins regulates their affinities for the ECM, whereas the binding of talin to actin constitutes the first link to the contractile machinery of cells. These 2 events are particularly important for cell migration. Indeed, this involves the cyclic attachment and detachment of integrins to the ECM but also the generation of a force required for the translocation of cellular contents (Atherton, P. and al., 2015. Vinculin controls talin engagement with the actomyosin machinery (Nat. Commun, Volume 6, p. 10).

[0169] The increase in the expression of the talin-1 gene combined with that of paxillin demonstrates a beneficial action on cell migration, particularly in the skin healing process.

[0170] PTGS2 Underexpression in an Anti-Inflammatory Context:

[0171] Prostaglandin-endoperoxide synthases, including cyclooxygenase-2 COX2 or PTGS2, catalyze the biosynthesis of prostaglandins (PG) from arachidonic acid in order to induce the inflammatory process via the secretion of cytokines and skin vasodilation.

[0172] The decrease in COX2 expression indicates the ability of the BCP active ingredient to reduce a possible inflammatory phenomenon that can be induced by different stimuli such as pathogens, UVs, ionizing radiation, etc.

[0173] I. Anti-Inflammatory Activity

[0174] The anti-inflammatory activity of the Bombax costatum polysaccharide extract according to Example 1 has been demonstrated with respect to an inflammation induced by PMA stress.

[0175] a) Materials and Methods

[0176] Normal Human Keratinocytes (NHK) were treated for 24 hours at 37° C. with the Bombax costatum Polysaccharide extract according to Example 1 (called BCP extract) at 0.01% and/or 0.05% of dry matter (DM) or by Dexamethasone or Indomethacin at 10-7M (anti-inflammatory reference molecules). The cells were then treated by adding PMA at 10 μg/ml (Phorbol Myristate Acetate, inflammation-inducing agent) for 16 hours at 37° C. At the end of the treatment, the quantities of IL1α (interleukin 1α), PGE2 (Prostaglandin-E2), IL1β (Interleukin 1β), TNFα (Tumor Necrosis Factor α), IL6 (Interleukin 6) and IL8 (Interleukin 8) secreted were measured by the ELISA technique in the culture supernatants.

[0177] The significance of the results was assessed by a one-way analysis of variance followed by a Tuckey test.

[0178] b) Results

[0179] As shown by the results in Tables 3 to 8, the BCP extract significantly inhibited the production of the inflammatory mediators IL1α, IL1β, IL6, IL8, TNFα and PGE2 in keratinocytes under inflammatory conditions.

[0180] These results show the anti-inflammatory activity of the BCP extract.

TABLE-US-00003 TABLE 3 Dosage of IL1-alpha produced by NHKs under the effect of PMA stress IL1α (pg/ml) Standard Average deviation Evolution Significance Control 33.869 2.340 PMA 10 μg/ml 372.604 91.220 +1000%  $$ Dexamethasone 0.1 μM 230.583 29.293 −38% ns BCP 0.05% dm 116.557 25.046 −69% * $$ p < 0.01 vs Control; * p < 0.05 **p < 0.01 vs PMA; ns Not Significant One-way ANOVA followed by a Tukey test

TABLE-US-00004 TABLE 4 Dosage of IL1-beta produced by NHKs under the effect of PMA stress IL1β (pg/ml) Standard Average deviation Evolution Significance Control 18.291 2.304 PMA 10 μg/ml 50.392 6.609 +175%  $$ Dexamethasone 0.1 μM 34.410 6.003 −32% ns BCP 0.05% dm 24.459 4.956 −51% * $$ p < 0.01 vs Control; * p < 0.05 vs PMA; ns Not Significant One-way ANOVA followed by a Tukey test

TABLE-US-00005 TABLE 5 Dosage of IL6 produced by NHKs under the effect of PMA stress IL6 (pg/ml) Standard Average deviation Evolution Significance Control 5.225 0.849 PMA 10 μg/ml 6.958 0.721 +33% $ Dexamethasone 0.1 μM 2.090 0.300 −70% *** BCP 0.01% dm 5.379 0.318 −23% * $ p < 0.05 vs Control; * p < 0.05 *** p < 0.001 vs PMA One-way ANOVA followed by a Tukey test

TABLE-US-00006 TABLE 6 Dosage of IL8 produced by NHKs under the effect of PMA stress IL8 (pg/ml) Standard Average deviation Evolution Significance Control 161.769 10.601 PMA 10 μg/ml 9952.388 1393.896 6052 $$$ Dexamethasone 0.1 4076.675 202.317 −59 *** μM BCP 0.01% dm 6641.050 804.243 −33 * $$$ p < 0.001 vs Control; * p < 0.05 *** p < 0.001 vs PMA; ns Not Significant One-way ANOVA followed by a Tukey test

TABLE-US-00007 TABLE 7 Dosage of TNF-alpha produced by NHKs under the effect of PMA stress TNFα (pg/ml) Standard Average deviation Evolution Significance Control 1.546 0.655 PMA 10 μg/ml 33.787 0.785 +2086%  $$$ Dexamethasone 0.1 μM 16.216 1.633 −52% *** BCP 0.01% dm 12.991 0.600 −62% *** BCP 0.05% dm 21.546 0.936 −36% * $$$ p < 0.001 vs Control; * p < 0.05 *** p < 0.001 vs PMA One-way ANOVA followed by a Tukey test

TABLE-US-00008 TABLE 8 Dosage of PGE2 produced by NHKs under the effect of PMA stress PGE2 (pg/ml) Standard Average deviation Evolution Significance Control 39.000 0.000 PMA 10 μg/ml 625.506 109.911 +1504%  $$$ Dexamethasone 0.1 μM 39.000 0.000 −94% *** BCP 0.05% dm 219.335 108.511 −65% ** $$$ p < 0.001 vs Control; ** p < 0.01 *** p < 0.001 vs PMA; ns Not Significant One-way ANOVA followed by a Tukey test

[0181] II. Activity on Epidermal Healing.

[0182] The potential activity on epidermal healing of the Bombax costatum Polysaccharide extract according to Example 1 (called BCP extract) was evaluated by studying keratinocyte migration, the first step in the process of skin re-epithelialization.

[0183] a) Materials and Methods

[0184] Scratch Assay: a lesion was performed on a mat of normal human keratinocyte monolayer (NHK) at confluence before treatment with BCP extract at 0.01% and 0.05% dry matter or with EGF (Epidermal Growth Factor) at 100 ng/ml.

[0185] A photo was taken at the start of treatment and after 5 hours of incubation in order to measure the rate of progression of the HCNs within the lesion. The percentage of coverage was evaluated under the different conditions by image analysis.

[0186] The significance of the results was assessed by Student's t test.

[0187] b) Results

[0188] As demonstrated by the results in Table 9, the BCP extract significantly stimulated keratinocyte migration, thus confirming its potential for activating skin healing.

TABLE-US-00009 TABLE 9 Migration of NHK Evolution compared % recovery to the control Control 33.6 ± 3.1  0 EGF 100 ng/ml 62.2 ± 11.1 +85% ** BCP 0.01% dm 55.4 ± 14.1 +65% *  BCP 0.05% dm 67.0 ± 8.6   +99% *** * p < 0.05 ** p < 0.01 *** p < 0.001 vs control; t test

[0189] III. Activity on Innate Anti-Microbial Defenses

[0190] The effect of the Bombax costatum Polysaccharide extract according to Example 1 (called BCP extract) was evaluated on the expression of anti-microbial peptides (AMPs) and of TLR2 in epidermal keratinocytes.

[0191] a) Materials and Methods

[0192] Normal human epidermal keratinocytes were treated for 24 or 48 hours with BCP extract or IL1β at 100 ng/ml (positive reference).

[0193] At the end of the treatment, the gene expression of hBD2, hBD3 and TLR2 was analyzed by real-time quantitative RT-PCR.

[0194] Moreover, the quantities of TLR2 and intracellular hBD2 were measured by ELISA.

[0195] b) Results

[0196] As demonstrated by the results in Tables 10 and 11, the BCP extract significantly stimulated the gene and protein expression of defensins and TLR2; thus demonstrating an anti-microbial activity and activation of immune defenses.

TABLE-US-00010 TABLE 10 Gene expression of PAMs and TLR2 in keratinocytes (RQ: Relative Quantity) hBD2 hBD3 TLR2 RQ Evolution RQ Evolution RQ Evolution Control 1.12 1.09 1.08 IL1β 100 ng/ml 3480.89 ×3000 *** 9.04 +730% *** 3.26  +201% *** BCP 0.01% dm 12.94   +1057% *** 2.69 +147% ns .sup.  2.14 +98% * BCP 0.05% dm 293.91  ×300 *** 10.69 +882% *** 2.81 +160% ** * p < 0.05; ** p < 0.01; *** p < 0.001; ns Not Significant vs Control One-way ANOVA followed by Dunnett test

TABLE-US-00011 TABLE 11 Production of hBD2 and TLR2 in keratinocytes (Intracellular protein expression) hBD2 TLR2 pg/ml Evolution pg/ml Evolution Control  141.5 ± 15.2 735.9 ± 39.2 IL1β 100 2162.7 ± 51.1 +1428% *** 913.7 ± 30.5 +24% ** ng/ml BCP 0.006% 286.33 ± 66.1 +102% *  789.2 ± 78.2 .sup. +7% ns dm BCP 0.02% dm  488.8 ± 57.3  +245% *** 884.2 ± 56.0 +20% *  * p < 0.05; ** p < 0.01; *** p < 0.001; ns Not Significant vs Control One-way ANOVA followed by Dunnett test

[0197] IV. Activity on Bacterial Adhesion

[0198] The effect of the BCP extract was evaluated on the adhesion of 4 bacterial strains to the surface of reconstructed human epidermis (RHE) or skin explants.

[0199] a) Materials and Methods [0200] Study of the adhesion of S. aureus, S. epidermidis and C. acnes:

[0201] Human Reconstructed Epidermis (RHE) were incubated for 15 min in the presence of the Bombax costatum polysaccharide extract according to Example 1 (called BCP extract) at 0.6% and 2%. Then, after two rinsings with PBS, the bacterial strains Staphylococcus epidermidis (ATCC 14990), Staphylococcus aureus (ATCC 6538) or Cutibacterium acnes (ATCC 6919) were deposited on the surface of the RHEs for 4 hours. After elimination of non-adherent bacteria by 4 successive washes, the RHEs were again incubated overnight.

[0202] The counting of the bacteria was carried out by counting the colonies after seeding on specific agar of the ground RHE. The result is expressed in CFU/RHE (colony forming unit). [0203] Study of the adhesion of C. xerosis:

[0204] Skin explants were incubated for 2 hours in the presence of BCP extract at 0.6% and 2%. A suspension of Corynebacterium xerosis (DSM 20743) was then deposited on the surface of the skin explants for 2 hours. After rinsing, these explants were again incubated for 24 hours.

[0205] The bacteria adhering to the surface of the skin explants were recovered by scraping then seeded on agar in order to carry out the count expressed in CFU/cm.sup.2.

[0206] b) Results

[0207] At the two concentrations tested, the BCP extract induced a marked inhibition of the adhesion of S. aureus on RHEs. At 2%, the BCP extract also inhibited the adhesion of C. acnes. (See table 12)

[0208] The BCP extract significantly inhibited the adhesion of C. xerosis at the surface of explants (see Table 13).

[0209] The BCP extract promotes a rebalancing of the skin microbiota by limiting the adhesion of pathogenic bacteria while preserving the adhesion of commensal bacteria.

TABLE-US-00012 TABLE 12 Bacterial adhesion on RHE C. acnes S. aureus S. epidermidis Evo- Evo- Evo- UFC/RHE lution UFC/RHE lution UFC/RHE lution Control 23125 1275000 2764801 BCP 0.6% NC NC 137875 −89% 2650852  −4% BCP 2%  8125 −65% 244750 −81% 2146464 −22%

TABLE-US-00013 TABLE 13 Adhesion of C. xerosis on skin explants UFC/cm.sup.2 Evolution Significance Control 597 ± 190 BCP 0.6% 272 ± 114 −54% ** BCP 2% 48 ± 38 −92% *** ** p < 0.01; *** p < 0.001 - one-way ANOVA followed by Dunnett test

[0210] V. Activity on Bacterial Growth

[0211] The antimicrobial or, on the contrary, prebiotic activity of the Bombax costatum polysaccharide extract according to Example 1 (called BCP extract) was evaluated by studying the bacterial growth of different strains cultured separately or in co-culture.

[0212] a) Materials and Methods

[0213] The following microbial strains, representative of the skin microbiota, were used: [0214] Staphylococcus aureus (ATCC 6538); [0215] Staphylococcus epidermidis (ATCC 12228); [0216] Cutibacterium acnes (ATCC 11827); [0217] Corynebacterium xerosis (ATCC 373); [0218] Malassezia furfur (ATCC 14521); [0219] Staphylococcus hominis (ATCC 27844). [0220] Determination of the minimum inhibitory concentration (MIC) and determination of the “activator” potential of bacterial growth:

[0221] The BCP extract was diluted in microplates at 0.25%; 0.5%; 1%; 2%; 4% and 8%, in the minimal culture medium specific to each strain. The controls were prepared similarly to the sample: [0222] A positive growth inhibition control: Phenonip® 5% in the diluted medium, [0223] A control consisting of diluted growth medium, [0224] A control consisting of growth medium.

[0225] The strains were added at the different conditions before incubation for 48 hours.

[0226] In the presence of a disorder revealing microbial growth after 48 hours, an aliquot was taken for the three highest concentrations still showing growth of microorganisms. These aliquots were deposited in separate plates in decimal dilutions, so as to determine the concentration of the populations by limiting dilutions.

[0227] After 48 hours of incubation, the activation of bacterial growth was demonstrated by determining the concentration of each microorganism (carried out simultaneously by visual reading and measurement of absorbance at 620 nm (turbidity)) according to decimal dilutions carried out.

[0228] Finally, the results were expressed by comparison with the bacterial growth obtained in the controls without product. [0229] Determination of the effect on the growth of bacteria in co-culture:

[0230] The S. aureus, S. epidermidis, S. hominis and C. acnes strains in co-culture were incubated for 48 hours in the presence of the 0.25% BCP extract; 0.5% and 1% or Phenonip 0.5%, positive growth inhibition control.

[0231] An aliquot was taken and placed on agars specific to the selected strains. After 48 hours of incubation, counting was performed for each strain.

[0232] b) Results [0233] Determination of the minimum inhibitory concentration (MIC) and determination of the “activator” potential of bacterial growth:

[0234] On C. acnes, an inhibition of bacterial growth was observed with a MIC determined at 4%. The growth kinetics showed a bacteriostatic effect of the BCP extract against C. acnes (see FIG. 1).

[0235] Similarly, the growth kinetics of M. furfur showed a bacteriostatic effect of the BCP extract against this yeast (see FIG. 1). [0236] Determination of the effect on the growth of bacteria in co-culture:

[0237] The BCP extract showed a nourishing effect against S. epidermidis, S. hominis and C. acnes strains and tended to inhibit the growth of S. aureus (bactericidal effect) (see FIG. 2).

[0238] These results show a protective effect on the balance of the skin microbiota.

[0239] VI. Defense Against S aureus Induced Damage

[0240] The bacterium Staphylococcus aureus (S. aureus) is frequently detected in patients with atopic dermatitis.

[0241] The quantity of S. aureus present in these patients is correlated with the degree of severity of the pathological condition and plays an important role in the pathophysiology.

[0242] The objective of this study was to reproduce the stress caused by this bacterium on the epidermis, for this purpose secretum of S. aureus was applied to the surface of RHE in order to study the consequences on proteins of epidermal differentiation.

[0243] a) Materials and Methods

[0244] The BCP extract according to Example 1 at 1% was applied as a 24-hour pre-treatment to the surface of reconstructed human epidermis (RHE). The RHEs were then treated topically by depositing the secretum (culture medium) of Staphylococcus aureus (ATCC 33592).

[0245] After an additional 24 hours of incubation, a morphological analysis of the tissues was carried out after Hematoxylin/Eosin staining, as well as the analysis of the expression of barrier function markers by immunofluorescence. The level of expression of the proteins of interest was evaluated by semi-quantification using Image J software.

[0246] b) Results

[0247] Morphological Analysis:

[0248] As shown in FIG. 3, the secretum of S. aureus induced a moderate alteration in the morphology of the RHE: disorganization of the structure of the epidermis (basal layer), fewer grains of keratohyaline at the granular layer.

[0249] The pre-treatment with the BCP extract preserved the morphology of the epidermis which are better organized, thicker and with a more marked production of keratohyaline grains.

[0250] Expression of Barrier Function Markers:

[0251] As demonstrated by the results in Table 14, S. aureus secretum significantly reduced the level of expression of the barrier function markers studied: Corneodesmosin, Desmoglein-1 and Filaggrin. This model is therefore representative of the negative impact of S. aureus on the barrier function, in particular in the pathophysiology of atopic dermatitis.

[0252] The BCP extract allowed to counterbalance this decrease in expression.

TABLE-US-00014 TABLE 14 Expression of barrier markers in RHEs Corneodesmosine Desmoglein1 Filaggrin Control 100   100    100   Secretum S. aureus 49.6 $ 35.4 $$  42.5 $$ Secretum + BCP 1% 84.2 * 120.1 **  79.4 * $ p < 0.05; $$ p < 0.01 vs Control; * p < 0.05; ** p < 0.01 vs secretum - Unpaired t test

[0253] The BCP extract could therefore protect the skin from aggression by S. aureus.

[0254] VII. Protective Activity of the Vaginal Microflora

[0255] The prebiotic efficacy of the BCP extract according to Example 1 was evaluated on 3 strains of Lactobacilli representative of the vaginal tract; moreover, the BCP extract was studied in a reconstructed model of vaginal epithelium colonized by strains of lactobacilli representative of the resident microflora.

a) Materials and Methods

[0256] Study of the prebiotic effect on L. gasseri, L. acidophilus, L. rhamnosus:

[0257] The L. gasseri (ATCC 33323), L. acidophilus (LA-14) and L. rhamnosus (ATCC 53103) strains were inoculated in a depleted medium in the presence of the Bombas costatum polysaccharide extract according to Example 1 (called BCP extract) at 0.25%; 0.5%; 1% and 2% or with addition of 2% glucose (positive control).

[0258] After 4 h and 24 h of incubation, the bacterial growth was evaluated by spectrophotometric measurement of the OD at 600 nm (which gives an indication of the rate of bacterial replication), as well as the bacterial viability expressed in CFU/ml (to quantify the number of viable residual bacteria).

[0259] The influence on bacterial metabolism was evaluated by measuring the production of lactic acid in the culture supernatants after 24 hours of incubation. [0260] Study of the protective effect on reconstituted vaginal epithelium:

[0261] The 1% BCP extract was applied to the surface of a reconstructed human vaginal epithelium (HVE), at the same time, the epithelia were colonized by a mixture of L. crispatus (DSM 20356) and L. gasseri (DSM 20243), mimicking the physiological resident microflora.

[0262] After 6 hours of incubation, the gene expression of the anti-microbial peptide hBD2 was evaluated by real-time RT-PCR.

[0263] b) Results [0264] Study of the prebiotic effect on L. gasseri, L. acidophilus, and L. rhamnosus

[0265] The bacterial growth rate evaluated by measuring the DO showed a positive effect of the BCP extract on the growth of L. gasseri, in a dose-dependent manner from 4 hours (early prebiotic effect); as well as on L. acidophilus, with more particularly a prebiotic effect after 24 hours of incubation (stationary phase) (see FIGS. 4a and 4b).

[0266] Overall, the BCP extract had a similar or even greater effect than the positive control (glucose) on the viability of lactobacilli, confirming its prebiotic effect.

[0267] Evaluation of bacterial viability (see Table 15) showed that the 2% BCP extract showed the greatest prebiotic effect at 4 hours on the 3 strains of lactobacilli, in particular L. gasseri and, to a lesser extent, L. rhamnosus. This tendecy continues up to 24 hours for L. rhamnosus, while a significant drop in viability was observed at 24 hours for L. gasseri, which is explained by starvation related to culture in an impoverished medium.

[0268] At 0.25% the BCP extract induced a prebiotic effect on L. acidophilus.

TABLE-US-00015 TABLE 15 Bacterial viability of lactobacillus strains L. gasseri L. acidophilus L. rhamnosus (Log10 CFU/ml) (Log10 CFU/ml) (Log10 CFU/ml) 4 h 24 h 4 h 24 h 4 h 24 h Control 7.98 ± 3.10 ± 7.77 ± 5.84 ± 7.82 ± 8.23 ± 0.01 0.28 0.13 0.09 0.21 0.00 Glucose 7.81 ± 3.42 ± 7.73 ± 7.22 ± 7.83 ± 9.09 ± 2% 0.27 0.14 0.23 0.15 0.06 0.07 BCP 7.89 ± 2.49 ± 7.72 ± 8.99 ± 7.77 ± 8.89 ± 0.25% 0.14 0.12* 0.13 0.55* 0.10 0.27 BCP 8.01 ± 0.00 ± 7.74 ± 6.10 ± 7.83 ± 8.51 ± 0.5% 0.19 0.00** 0.08 0.57 0.06 0.56 BCP 8.02 ± 3.27 ± 8.10 ± 6.29 ± 7.74 ± 7.82 ± 1% 0.10 0.38 0.58 0.02 0.06 0.01* BCP 10.40 ± 3.73 ± 8.83 ± 6.63 ± 8.75 ± 10.35 ± 2% 0.24** 0.07 0.18 0.25 0.11** 0.04* *p < 0.05; **p < 0.01 - ANOVA test

[0269] Lactic acid, a product of the primary metabolism of lactobacilli, was quantified in L. Acidophilus culture media (see Table 16).

[0270] The BCP extract induced an increase in lactic acid production by L. acidophilus.

TABLE-US-00016 TABLE 16 Quantification of lactic acid produced by L. acidophilus L. acidophilus Lactic Acid (nmol/μl) Evolution Control 25421.81 ± 4389.106 Glucose 2% 24200.96 ± 1062.115  −5% BCP 0.25% 57556.58 ± 29969.59 +126% BCP 0.5% 46584.36 ± 2749.858  +83% BCP 1% 48314.47 ± 12267.67  +90% BCP 2% 90768.17 ± 945.7198 +257% *p < 0.05; **p < 0.01 - ANOVA test

[0271] Overall, these results show a prebiotic effect of the BCP extract against L. acidophilus. This effect is greater at a concentration of 2%.

TABLE-US-00017 TABLE 17 Summary of the prebiotic effect on lactobacilli Growth rate Viability Metabolism 4 h 24 h 4 h 24 h (Lactic acid) (exponen- (station- (exponen- (station- 24 h tial ary tial ary (station- phase) phase) phase) phase) ary phase) L. gasseri ++ + ++ + + L. + ++ + ++ ++ acidophilus ++ greater effect than positive control; high prebiotic effect + effect equivalent to the positive control; prebiotic effect − no change compared to the control; lack of prebiotic effect [0272] Study of the protective effect on reconstituted vaginal epithelium (HVE):

[0273] In the colonized vaginal epithelium (HVE) model, the BCP extract significantly increased hBD2 gene expression (see Table 18).

[0274] This result shows a booster effect of local epithelial defenses to protect the mucous membrane from the risk of infection.

TABLE-US-00018 TABLE 18 Gene expression of hBD2 in HVE colonized by Lactobacilli hBD2 (RQ) Control 1.00 HVE colonized 0.88 HVE colonized + BCP 1% 3.24 RQ: Relative Quantity

[0275] VIII. In Vitro Sniff Test (Deodorant Activity)

[0276] An in vitro “sniff test” was carried out in order to evaluate the ability of the BCP extract according to Example 1 to inhibit or modify the production of odors by the main bacterial species responsible for underarm odors: C. striatum, S. epidermidis.

[0277] a) Materials and Methods

[0278] Staphylococcus epidermidis (ATCC 12228) and Corynebacterium striatum (ATCC 6940) strains were used.

[0279] Each of these strains has a typical olfactory signature, the result of the metabolism of sweat components: [0280] S. epidermidis: Valeric and propionic acid; [0281] C. striatum: Sulphanyl alcohols.

[0282] Each bacterial strain was inoculated into a reconstituted sweat solution in the presence of the BCP extract at 0.1%; 0.6% or 2% or 0.1% chlorhexidine digluconate (positive control).

[0283] After 6 h and 24 h of incubation:

[0284] The bacterial viability was evaluated by bacterial count expressed in CFU/ml.

[0285] A sniff test was carried out to determine the olfactory signature qualitatively and semi-quantitatively (intensity of the smell) immediately after opening the vial, the smell was evaluated and scored by a trained operator: [0286] 1. Absent (comparable to 0.1% chlorhexidine positive control) [0287] 2. Moderate [0288] 3. Intense (bacteria-specific olfactory signature)

[0289] b) Results

[0290] The BCP extract inhibited the odor produced by S. epidermidis and C. striatum compared to the negative control, without altering the viability of these bacteria (see Table 19). These results demonstrate deodorant activity.

TABLE-US-00019 TABLE 19 Viability and odor of bacteria in reconstituted sweat 6 h 24 h Viability Odor Viability Odor (Log10 CFU/ml) (Sniff) (Log10 CFU/ml) (Sniff) S. epidermidis Control 8.91 ± 0.21 Intense 8.69 ± 0.09 Intense (reconstituted sweat) Chlorhexidine   0.00 ± 0.00 ** Absent   0.00 ± 0.00 ** Absent BCP 0.1% 8.80 ± 0.08 Moderate 8.64 ± 0.30 Moderate- Intense BCP 0.6% 8.87 ± 0.07 Moderate- 8.87 ± 0.15 Moderate- Absent Absent BCP 2% 8.73 ± 0.34 Moderate 8.78 ± 0.18 Moderate- Absent C. striatum Control 8.51 ± 0.08 Moderate 8.64 ± 0.19 Moderate (reconstituted sweat) Chlorhexidine   0.00 ± 0.00 ** Absent   0.00 ± 0.00 ** Intense BCP 0.1% 8.59 ± 0.31 Moderate 8.38 ± 0.11 Moderate BCP 0.6% 8.66 ± 0.32 Absent 8.57 ± 0.11 Moderate- Absent BCP 2% 8.28 ± 0.06 Absent 8.31 ± 0.28 Absent ** p < 0.01 - ANOVA test vs Control

[0291] IX. Study of Bacterial Biofilm

[0292] A biofilm is defined as the assembly of microbial cells associated with a living organism or a tissue and “coated” in a polysaccharide matrix.

[0293] The biofilm is one of the most important virulence factors in infectious diseases, it confers resistance to antibiotics, protection against host defenses, it increases the virulence of pathogenic bacteria by promoting their communication system (Quorum Sensing).

[0294] In the skin, biofilms have been described in various pathological conditions or disorders: acne, rosacea, atopic dermatitis. Biofilms are also likely to disrupt healing mechanisms. Bacteria that associate in biofilm have an innate resistance to antibiotics, disinfectants and host defense systems; biofilms thus promote the persistence of pathogenic bacteria and their recalcitrance to treatment.

[0295] For example, the involvement of the Staphylococcus aureus biofilm has been particularly described in the context of the pathophysiology of atopic dermatitis. Indeed, probably due to the alteration of the surface of the skin which allows it to adhere, s. aureus is more easily found in the form of a biofilm in atopic skin; this state of biofilm promotes its resistance to treatment and increases its virulence, which results in the induction of chronic inflammation and pruritus, phenomena that contribute to the vicious circle of the pathogenesis of atopic dermatitis.

[0296] The effect of the BCP extract according to the present invention was evaluated on the biofilm-forming capacity of bacteria of the skin microflora.

[0297] a) Materials and Methods [0298] Microbial strains tested: [0299] Staphylococcus epidermidis MFP04: healthy human skin strain (LMSM set of strains) characterized by metabolic analysis, 16S RNA and genomic sequencing. It is a strain rather described as commensal. [0300] Staphylococcus aureus MFP03: healthy human skin strain (LMSM set of strains) characterized by metabolic analysis, 16S RNA and genomic sequencing. It is a commensal strain, an “opportunistic” pathogen, which can be responsible for certain skin infections and widely described as being involved in the pathophysiology of atopic dermatitis. [0301] Corynebacterium xerosis CIP 100653T/ATCC373: strain from international databases. It is a commensal strain, involved in the production of underarm odors. [0302] Cutibacterium (propionibacterium) acnes HL045PA1/HM-516 ribotype 4 (RT4): “acneic” strain characterized by Fitz-Gibbon and al. and obtained from an international database. It is a pathogenic strain, involved in the pathogenesis of acne. [0303] Cutibacterium (propionibacterium) acnes HL110PA3/HM-554 ribotype 6 (RT6): “non-acneic” strain characterized by Fitz-Gibbon and al. and obtained from an international database. It is a commensal strain. [0304] Micrococcus luteus 0116: healthy human skin strain (LMSM set of strains) characterized by metabolic analysis, 16S RNA and genomic sequencing. It is a commensal strain. [0305] Pre-study of the effect of the BCP active ingredient on bacterial growth kinetics:

[0306] A control study was carried out to verify the absence of effect of the BCP active ingredient on the growth of the 6 strains studied.

[0307] The active ingredient diluted to 1/50.sup.th was added to the medium from the start of the culture. Bacterial growth was monitored in microplates over 24 h, 48 h or 72 h depending on the strains. Absorbance was measured continuously using a microplate reader/incubator. [0308] Study of the effect of the BCP active ingredient on the formation of biofilm by the crystal violet technique:

[0309] The production of biofilm of bacterial species was studied in multi-well plates (96) using the crystal violet staining technique after culture in the presence of the active ingredient for 24, 48 or 72 hours depending on the strains.

[0310] The results are expressed as a percentage based on the biofilm formation value in the control medium (without active ingredient). Statistical differences were established using the Mann-Whitney test.

[0311] b) Results

[0312] The BCP extract of kapok polysaccharides according to the invention did not significantly modify the kinetics of bacterial growth under the conditions of the test.

[0313] The study of biofilm production by the different bacterial strains revealed the following effects of the kapok tree polysaccharide extract (see FIG. 5): [0314] significant inhibition of S. aureus biofilm formation; [0315] an induction of S. epidermis biofilm formation; [0316] an absence of significant modulation of the formation of biofilms of M. luteus, C. xerosis or C. acnes RT6 (non-acne strain); [0317] inhibition of the formation of the biofilm of the acne strain C. acnes RT4 (acne strain).

[0318] Advantageously, the BCP extract of kapok polysaccharides inhibits the biofilm formation capacity of pathogenic bacterial strains (S. aureus and C. acnes RT6 in particular), while preserving or even promoting the capacity of commensal bacteria to form a biofilm. These results are in favor of the effect of the active ingredient on the preservation of the balance of the microbiota, with in particular a benefit in pathological conditions such as atopic dermatitis or acne.