Compositions containing galactinol and uses thereof.

Abstract

The present invention relates to compositions comprising or consisting of 3-O-α-D-galactopyranosyl-D-myo-inositol as an active ingredient, as well as to a method for the biocatalytic production of galactinol having the formula 3-O-α-D-galactopyranosyl-D-myo-inositol.Furthermore, the present invention discloses the cosmetic use of a composition comprising or consisting of 3-O-α-D-galactopyranosyl-D-myo-inositol for maintaining and / or restoring healthy skin through the stimulation of its own endogenous defense mechanisms, for inducing the production of extracellular matrix components in skin cells, for stimulating growth factor production, for maintaining and / or enhancing the skin microbiota, and more generally for improving the overall skin homeostasis and the overall skin appearance.

Description

[Technical field]

[0001] The present invention relates to compositions comprising 3-O-α-D-galactopyranosyl-D-myo-inositol (galactinol), its use as an active ingredient, in particular in the cosmetic field, as well as to methods for producing such compositions.The present invention also relates to the cosmetic use of 3-O-α-D-galactopyranosyl-D-myo-inositol as an active ingredient in skin care compositions. [Background technology]

[0002] At the interface between the organism and the environment, the skin - composed of three distinct layers: the stratum corneum (hereafter referred to as "SC"), the epidermis and the dermis - provides mechanical protection and plays the role of the first barrier against external aggressions and against uncontrolled loss of body fluids. External aggressions are, for example, ultraviolet and infrared exposure, particulate matter pollution, pathogens, allergens, temperature and humidity fluctuations, chemical products, mechanical restraints due to daily habits, such as shaving, gloves and wearing a mask due to the hygienic decay of our environment due to the epidemic of harmful viruses. In addition, the skin also protects the organism from internal aggressions, such as chronological aging, hormonal fluctuations and immune deregulation. For example, intrinsic and extrinsic aging processes are known to be associated with phenotypic changes in skin cells, with major structural and functional changes occurring in the dermal extracellular matrix (ECM), the connective tissue that surrounds the cells, containing fibers and substrates composed by water and macromolecules (e.g. proteoglycans). Indeed, fibrous collagen, elastic fibers (e.g., elastin (ELN)) and proteoglycans are necessary to provide tensile strength, elasticity (recoil), resilience and hydration to the skin, respectively. In this regard, collagen (COL) provides tensile strength to the skin. Collectively, glycoproteins and proteoglycans, together with the abundant polymeric hyaluronic acid (HA), are distributed throughout the skin, where they play an important role in maintaining skin hydration. Furthermore, proteoglycans are present in many connective tissues, on the cell surface, and in intracellular compartments.

[0003] Proteoglycans, such as decorin (DCN), perlecan (PLC), lumican (LUM), syndecan (SDC) and versican (VCAN), a family of glycosaminoglycan (GAG)-conjugated proteins, are important components of human skin connective tissue (e.g., dermis) and are known to be essential for maintaining the mechanical strength of the skin (Martinez et al., “Role of proteoglycans on skin ageing: a review”. International Journal of Cosmetic Science, Vol.42; p.529-535, 2020: Non-Patent Document 1).

[0004] In fact, these proteoglycans are essential components that repair cells, provide hydration, maintain the hydration of the ECM, prevent the formation of wrinkles thanks to their ability to bind other molecules such as collagen or hyaluronic acid, and favor the smoothness of the skin texture. For example, hyaluronic acid is known to help retain more than 1,000 times its weight of water within skin cells, making it an excellent moisturizer. The dermis contains up to 50% of the internal hyaluronic acid. Moreover, apart from their hydrodynamic function, proteoglycans have well-defined biological functions. For example, decorin, found in the dermis, regulates collagen fibrillogenesis and modifies the activity of transforming growth factor-β.

[0005] Furthermore, all the aforementioned attacks are known to induce many deleterious effects on the cell, but also on the molecular level, e.g.: - Generation of reactive oxygen species (ROS). ROS cause damage to numerous cellular organelles and processes, which ultimately disrupt normal cellular physiology. Fortunately, a delicate balance exists between ROS production and antioxidant defenses that protect cells in vivo. Indeed, cells have deployed various antioxidant defense mechanisms, such as the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway, detoxifying enzymes (e.g. superoxide dismutase (SOD), glutathione peroxidase-1 (GPX1), NAD(H) dehydrogenase quinone 1 (NQO1) and catalase (CAT)), that can neutralize and / or metabolize these harmful ROS; - Proteome alterations due to protein denaturation, aggregation, and misfolding, which can be counteracted by cellular defenses such as heat shock proteins (HSPs) and chaperone proteins involved in proteome integrity and repair. In particular, the mammalian HSP90 family of proteins, a cluster of highly conserved molecules involved in a myriad of cellular processes, is essential for cellular homeostasis. Furthermore, HSP27 plays an important role in skin homeostasis as hydration, due to the regulation of the final step of keratinization in the stratum corneum. Furthermore, HSP27 has been described to be involved in filaggrin (FLG), loricrin (LOR) and keratin 10 (KRT10) production. - DNA damage, e.g. mutations, deaminations, missing bases, chemical modifications of bases, formation of pyrimidine dimers (e.g. thymine dimers) and strand breaks, can be countered by various DNA repair enzymes produced by skin cells, e.g. growth arrest and DNA damage inducible protein alpha (GADD45-α), proliferating cell nuclear antigen (PCNA). DNA maintenance is known to be an important mechanism limiting the effects of senescence, aging and apoptosis on cells and organs. - A decrease in the rate of cell division in self-renewing tissues such as the skin (decreased PCNA and Ki67 proliferation markers) leading to a reduced ability to maintain skin homeostasis.

[0006] Moreover, when these attacks are persistent and excessive, they alter the skin's self-defense and self-repair capabilities. In fact, the skin is no longer able to induce its own endogenous defense mechanisms, such as its antioxidant mechanisms, heat shock proteins, DNA repair enzymes, its proteomic protection by growth factor production, nor is it able to stimulate the production of its main ECM components. As a result, the skin becomes more fragile, dehydrated, unhealthy and prone to premature aging.

[0007] Keratinocytes and fibroblasts are known to be found widely in both the skin and hair follicles.

[0008] Recently, the term "metaproteomic" has emerged to describe the most abundantly expressed genes and / or proteins in environmental samples. More precisely, the term "metaproteomic" refers to the proteomic analysis of a mixture of species, which uses a suitable MS to generate mass spectrometer (MS) data and searches the MS data with a compilation of protein sequence databases that represent at least a portion of the species in the mixture. Exemplary methods of metaproteomic analysis are known (Xiong Weili et al., Journal of Proteome Research., 14, 1, 133-141, 2014). Metaproteomics is a tool for the large-scale analysis of proteins in the microbiome that allows researchers to address diverse questions related to functions and interactions in the microbiome and the host.

[0009] It is agreed that the crosstalk between the host and its microbiome is an essential part of many biological functions and that maintaining a precise balance between these two components of the skin is essential for human health.

[0010] Galactinol (3-O-α-D-galactopyranosyl-D-myo-inositol) is a natural molecule widely present among various plant species and is used as a galactose donor for the synthesis of the raffinose family of oligosaccharides (RFO's).

[0011] Galactinol is a disaccharide composed of myo-inositol linked to galactose. This nomenclature encompasses several isomers. Thus, there are multiple galactinol isomers, depending on both the bond between the two sugars and the configuration of each sugar.

[0012] The biosynthesis of galactinol from inositol and uridine diphosphate-galactose is known to be a key step in the synthesis of RFO's and is catalyzed by the enzyme galactinol synthase (GolS). In plants, the expression of GolS is induced by abiotic stress and as such triggers the synthesis of RFO's as a means to protect the plant from drought and other types of stress, such as salt and cold.

[0013] In plants, galactinol exists in very small amounts, and it is difficult to produce it in sufficient quantities to meet the needs in the cosmetic field.For example, small amounts of galactinol can be isolated from sugar beet syrup or castor oilseed meal (McCready et al., Journal of the ASSBT: 127-132, 1965: Non-Patent Document 3).Galactinol isomer (1-O-α-D-galactopyranosyl-myo-inositol) can also be obtained from biocatalytic synthesis from three substrates (sucrose, uridine diphosphate (UDP) and myo-inositol) by using three enzymes (sucrose synthase, UDP-galactose-4-epimerase and galactinol synthase) (Wakiuchi et al., Bioscience, biotechnology and biochemistry 67 (7): 1465-1471, 2003: Non-Patent Document 4). Using this biocatalytic synthesis, a single product is obtained in which myo-inositol is selectively galactosylated to give a product identical to galactinol isolated from soybean (Schweizer et al., Carbohydrates Research, 95, 61-71, 1981).

[0014] Galactosylation can generally be carried out by transglycosylation, for example, where a galactose donor such as melibiose is used for the galactosylation of tyrosol (Potocka et al., Journal of Molecular Catalysis B: Enzymatic 113, 23-28, 2015: Non-Patent Document 6).

[0015] The synthesis of galactinol can be applied to the biocatalytic synthesis of soybean oligosaccharides, such as raffinose and stachyose, as described in the document CN-A107586809 (Patent Document 1). It is described therein that raffinose synthetase and stachyose synthetase, which catalyze the conversion of sucrose to raffinose and raffinose to stachyose, respectively, are utilized with galactinol as the galactose donor. However, this biocatalytic synthesis requires a total of three different enzymes to perform the total synthesis of galactinol.

[0016] Galactinol isomers are already used in the cosmetics field.

[0017] For example, JP-A2007084484 (Patent Document 2) describes inositol glycosides containing myo-inositol (galactinol) linked to galactose for alleviating cell damage caused by UV exposure, thus in preventing pigmentation. Among the galactinol isomers, the β-isomer is disclosed. Galactinol isomers with 1-O-α-D, 2-O-α-D, 4-O-α-D and 5-O-α-D-galactopyranosyl-myo-inositol as linkages are also described. However, 3-O-α-D-galactopyranosyl-D-myo-inositol is missing.

[0018] Document US-B2 10918585 (Patent Document 3) teaches a skin anti-aging composition consisting of an inositol derivative in which glucose or an oligosaccharide containing glucose as a building block is bound to myo-inositol. However, galactose is not disclosed as an oligosaccharide.

[0019] Moreover, the inositol derivatives are also used as agents for suppressing skin troubles caused by fine particle substances (EP-A1 3636266: Patent Document 4).

[0020] Document EP-B1 1744725 discloses myo-inositol combined with glucose for improving skin moisture.

[0021] Document JP-B2 4624831 (Patent Document 6) describes external skin preparations containing inositol bound to sugars for improving skin moisture and smoothness. Galactose is not disclosed as a sugar.

[0022] The aforementioned galactinol isomers have several technical drawbacks. In many cases, their production by biosynthesis is costly, since it involves several enzymes and substrates in multiple enzyme cascades and generates large amounts of unusable by-products. Moreover, each of these enzymes and substrates can be expensive, which will undoubtedly lead to a high cost of production of the final product (galactinol), which itself is expensive. Another technical drawback is that all the enzymes disclosed for the biocatalytic synthesis of galactinol are plant-derived enzymes, which themselves can sometimes be difficult to express in prokaryotic expression hosts. Finally, to the best of our knowledge, the production of galactinol in high yields is not sufficient to meet the needs of cosmetic applications. [Prior art documents] [Patent documents]

[0023] [Patent Document 1] CN-A107586809 [Patent Document 2] JP-A2007084484 [Patent Document 3] US-B2 10918585 [Patent Document 4] EP-A1 3636266 [Patent Document 5] EP-B1 1744725 [Patent Document 6] JP-B2 4624831 [Non-patent literature]

[0024] [Non-Patent Document 1] Martinez et al., “Role of proteoglycans on skin aging: a review”. International Journal of Cosmetic Science, Vol.42; p.529-535, 2020 [Non-Patent Document 2] Xiong Weili et al., Journal of Proteome Research., 14, 1, 133-141, 2014 [Non-Patent Document 3] McCready et al., Journal of the ASSBT: 127-132, 1965 [Non-Patent Document 4] Wakiuchi et al., Bioscience, biotechnology and biochemistry 67 (7): 1465-1471, 2003 [Non-Patent Document 5] Schweizer et al., Carbohydrates Research, 95, 61-71, 1981 [Non-Patent Document 6] Potocka et al., Journal of Molecular Catalysis B: Enzymatic 113, 23-28, 2015 Summary of the Invention [Problem to be solved by the invention]

[0025] Thus, there is still an unmet need for the production of galactinol for use in the cosmetic field.Furthermore, it is desirable for the galactinol to be derived from natural sources.

[0026] There is also an unmet need for more efficient biocatalytic syntheses to produce native galactinol isomers at lower cost by using melibiose or another substrate as the galactose donor. Thus, further, the yields of galactinol produced by known biocatalytic syntheses are low, and there is also an unmet need to provide a method for the large-scale production of galactinol with limited amounts of its isomers and / or its isoforms.

[0027] It is also desirable to maintain healthy skin by protecting it from harmful agents, and it is also desirable to have skin that exhibits good physiological and physical properties and thus has an overall good appearance. [Means for solving the problem]

[0028] These objectives can be achieved by compositions of natural origin that are able to mobilize and / or activate in healthy skin its own endogenous defense mechanisms (e.g. antioxidant mechanisms, protection of its proteome, DNA repair enzymes, production of growth factors) and preserve its microbiota sources, so that the skin can become more remodeled, hydrated, protected, healthy and more resilient against both premature and pro-aging processes, and more generally, the skin presents a younger and healthier overall appearance.

[0029] Summary of the Invention Surprisingly, it has been found that the "natural" galactinol compound corresponding to the formula 3-O-α-D-galactopyranosyl-D-myo-inositol can be obtained by a new biocatalytic synthesis. As a result, it has also been found that myo-inositol can be galactosylated efficiently in high yield.

[0030] The present invention relates to a composition comprising 3-O-α-D-galactopyranosyl-D-myo-inositol obtainable by biocatalytic synthesis starting from melibiose as galactose donor.

[0031] Thus, one aspect of the present invention relates to a composition comprising 3-O-α-D-galactopyranosyl-D-myo-inositol as an active ingredient.

[0032] Another aspect of the present invention relates to a biocatalytic process for the production of galactinol having the formula 3-O-α-D-galactopyranosyl-D-myo-inositol, comprising the steps of: a.1) optionally providing melibiose or incubating raffinose with β-fructofuranosidase at a pH between 3 and 8 to obtain melibiose; and incubating a mixture comprising melibiose and myo-inositol with α-galactosidase at a pH between 4 and 9; or a.2) if step a1) was not performed, incubating a mixture comprising myo-inositol and raffinose and / or stachyose with α-galactosidase at a pH between 4 and 9; b. Purifying 3-O-α-D-galactopyranosyl-D-myo-inositol from the mixture of compounds obtained in step a1) or step a2).

[0033] Another aspect of the present invention is a method for producing a composition comprising the steps of: a. 3-O-α-D-galactopyranosyl-D-myo-inositol as the active ingredient; and b. at least one additional cosmetically and / or pharma- ceutically acceptable ingredient other than 3-O-α-D-galactopyranosyl-D-myo-inositol; A cosmetic or pharmaceutical composition comprising: The cosmetic or pharmaceutical composition is a composition for topical use selected from the group consisting of a solution, a suspension, an emulsion, a cream, a paste, a gel, a lotion, a powder, a soap, a water containing a surfactant, an oil, a shampoo and a spray; and / or is a nutraceutical composition administered orally.

[0034] Another aspect of the present invention relates to the cosmetic use of a composition comprising 3-O-α-D-galactopyranosyl-D-myo-inositol for maintaining and / or restoring healthy skin through stimulation of its own endogenous defense mechanisms, for inducing the production of extracellular matrix components in skin cells, for stimulating growth factor production and more generally for improving overall skin homeostasis and overall skin appearance.

[0035] A further aspect of the present invention relates to a composition comprising (or consisting of) 3-O-α-D-galactopyranosyl-D-myo-inositol as an active ingredient for use in a non-therapeutic method for maintaining and / or improving the overall appearance of the skin and its annexes, said composition being for maintaining or improving skin homeostasis, for maintaining healthy skin, for improving skin firmness and / or elasticity, for protecting the skin against external stressors, for improving skin relief, for improving skin hydration and skin barrier function, for preventing and / or delaying the signs of skin ageing, and with regard to already aged skin, said composition being for protecting and enhancing the mobilization of its microbiota sources.

[0036] The experimental section of this application demonstrates that the composition of the present invention can be used to improve the overall appearance of the skin and its appendages, which can advantageously upregulate the expression of key genes, important proteins, proteoglycans and glycosaminoglycans involved in achieving this goal. [Brief description of the drawings]

[0037] FIG. 1 shows the chemical structure of galactinol according to the present invention.

[0038] 2 shows the ability of galactinol and compositions containing it according to an embodiment of the present invention to stimulate gene expression of extracellular matrix (ECM) components in normal human dermal fibroblasts (NHDFs). The set of modulated genes includes collagen type V alpha 1 chain (COL5A1); elastin (ELN), laminin subunit gamma 2 (LAMC2), lumican (LUM), syndecan-4 (SDC4), and versican core protein (VCAN).

[0039] 3 shows the ability of galactinol and compositions containing it according to an embodiment of the present invention to stimulate antioxidant enzymes in NHDF. The set of modulated genes includes NAD(H) dehydrogenase quinone 1 (NQO1), heme oxygenase 1 (HMOX1), glutathione peroxidase 1 (GPX1) and glucose-6 phosphate dehydrogenase (G6PD).

[0040] FIG. 4 demonstrates the ability of galactinol and compositions comprising same according to an embodiment of the present invention to stimulate in NHDF the mRNA expression of key genes involved in maintaining overall skin homeostasis (i.e. protective proteins (heat shock proteins such as heat shock protein family A member A (HSPA1A) and heat shock protein 90 alpha family class A (HSP90AA)); DNA repair and cell proliferation: proliferating cell nuclear antigen (PCNA), proliferation marker Ki-67 (MKi67) and growth arrest and DNA damage 45-alpha (GADD45A); and growth factors such as transforming growth factor beta-1 (TGFβ1) and nerve growth factor (NGF)).

[0041] FIG. 5 demonstrates the ability of galactinol and compositions containing it according to an embodiment of the present invention to protect the proteome of NHDF by stimulating in these cells the significant expression of HSP27.

[0042] FIG. 6 shows the ability of a composition comprising galactinol according to the present invention to stimulate the production of hyaluronic acid (HA) in NHEKs.

[0043] FIG. 7 shows, at a clinical level, on human volunteers, the ability of a composition containing galactinol according to the invention to improve the biomechanical properties of the skin (by increasing the Ue and Ur parameters), to reduce skin wrinkles and to reduce skin microrelief (smoothing effect).

[0044] Detailed Description of the Invention One aspect of the present invention relates to a composition comprising (or consisting of) 3-O-α-D-galactopyranosyl-D-myo-inositol as an active ingredient.

[0045] In a composition comprising 3-O-α-D-galactopyranosyl-D-myo-inositol, it will be understood that the composition is 3-O-α-D-galactopyranosyl-D-myo-inositol.

[0046] As used throughout this disclosure, 3-O-α-D-galactopyranosyl-D-myo-inositol of the present invention is also referred to as “galactinol” [Cas number: 3687-64-7].

[0047] In one embodiment, the composition of the present invention can further comprise galactinol isomers with α-linkages other than 3-α-linkages.Advantageously, the galactinol isomers with α-linkages other than 3-α-D can be selected from 1-O-α-D-, 2-O-α-D-, 4-O-α-D-, 5-O-α-D or 6-O-α-D-galactopyranosyl-D-myo-inositol.The molar ratio between galactinol:galactinol isomers in the composition of the present invention can be approximately comprised between 3:1, preferably 2:1, in particular 1.5:1.

[0048] In a preferred embodiment, galactinol and the mixture comprising said galactinol isomers represent 20% by weight, preferably 25% by weight and in particular 30% by weight relative to the total weight of the composition.

[0049] In a preferred embodiment, the composition of the present invention may further comprise melibiose, myo-inositol, a monosaccharide or a mixture thereof, said monosaccharide being selected from galactose, glucose, fructose or a mixture thereof.

[0050] In a preferred embodiment, the composition of the invention may further comprise, relative to the total weight of the composition: - 0 to 25% by weight, preferably 0.1 to 20% by weight, of melibiose, - 0 to 40% by weight, preferably 0.1 to 35% by weight, of myo-inositol, - 0 to 30% by weight, preferably 0.1 to 25% by weight, of monosaccharides.

[0051] Advantageously, the compositions of the invention may contain galactinol as described above, for example in admixture with one or more of the compounds described above. Preferably, the relative amounts of each of these compounds in the compositions of the invention may be within the ranges described above.

[0052] 1. A biocatalytic process for the preparation of galactinol having the formula 3-O-α-D-galactopyranosyl-D-myo-inositol, comprising the steps of: A1) providing melibiose or incubating raffinose with β-fructofuranosidase at a pH between 3 and 8 to obtain melibiose; and incubating a mixture comprising melibiose and an acceptor selected from pinitol and / or inositol isomers including myo-, chiro-, scyllo-, chiro-, neo-, allo-, epi- and cis-inositol, preferably myo-inositol, with α-galactosidase at a pH between 4 and 9; or A2) if step A1) was not performed, incubating a mixture comprising an acceptor selected from pinitol and / or inositol isomers including myo-, chiro-, scyllo-, chiro-, neo-, allo-, epi- and cis-inositol, preferably myo-inositol, and raffinose and / or stachyose with α-galactosidase at a pH between 3 and 9; B) Purifying 3-O-α-D-galactopyranosyl-D-myo-inositol from the mixture of compounds obtained in step A1) or step A2).

[0053] It is therefore an object of the present invention to provide a method for utilizing abundant galactose donors by using enzymes capable of α-transglycosylation primarily at the 3-position of myo-inositol.

[0054] Advantageously, raffinose is a naturally abundant and inexpensive natural compound.For example, raffinose can be isolated from plants.Preferably, raffinose can be isolated from cottonseed, soybean seed, or legumes, such as, but not limited to, cabbage, broccoli, or asparagus.

[0055] It is worth noting that raffinose, melibiose and myo-inositol may be in solid form (e.g., powder) prior to the incubation step with the enzyme. Thus, the substrate donors may be solubilized in a suitable solvent and / or buffer during incubation with the enzyme. Thus, in one embodiment, raffinose may be dissolved in a buffer prior to the incubation step with β-fructofuranosidase. In a preferred embodiment, raffinose is dissolved in a sodium phosphate buffer at a pH of 4-8, preferably, the pH is in the range of 4.5-7, more preferably, the pH is 4.9-6.5.

[0056] As used herein, "β-fructofuranosidase", also called invertase, refers not only to the enzyme of EC 3.2.1.26 class, but also to molecules or functional equivalents thereof that exhibit the same properties with respect to their substrates and products. As used herein, "functional equivalents" or analogs of an enzyme are its various polypeptides, which further perform the desired biological function and / or activity, such as enzymatic activity. Advantageously, it is an enzyme of yeast origin that can specifically hydrolyze the terminal non-reducing β-D-fructofuranosidic residue in β-D-fructofuranosides.

[0057] The term "α-galactosidase", also called melibiase, as used herein, refers not only to enzymes of the EC 3.2.1.22 class, but also to molecules or their functional equivalents that exhibit the same properties with respect to their substrates and products. Advantageously, these enzymes of microbial origin are able to specifically hydrolyze terminal non-reduced α-D-galactosyl moieties from a wide variety of products, for example glycoproteins, glycolipids or oligosaccharides, including galactose oligosaccharides, galactomannans and galactolipids. Apart from these standard reactions, these enzymes are also able to catalyze the formation of α-galactosyl bonds. This can be done in a "reverse hydrolysis" manner or by using an activated galactose donor. In the first case, an equilibrium is reached between the reagents (galactose and acceptor) and the products (galactosylated product and water). The position of this equilibrium is to the left and in many cases high concentrations of galactose are required even to obtain low conversions.

[0058] According to the invention, the preferred acceptor is myo-inositol. Other inositol isomers, such as chiro-, scyllo-, chiro-, neo-, allo-, epi- and cis-inositol, or inositol derivatives, such as pinitol, can be used, since they also act as acceptors.

[0059] In a preferred embodiment, the composition comprises, based on the total weight of the composition: - 0-25% by weight of melibiose, - 0-40% by weight of myo-inositol, - Monosaccharides 0-30% by weight Includes.

[0060] Higher conversions can be obtained using activated galactose donors in the transglycosylation reaction; typical α-galactose donors are p-nitrophenol-α-galactose and melibiose. The latter has the advantage of being a relatively abundant natural molecule. However, although melibiose is commercially available, it is very expensive compared to, for example, raffinose. Raffinose and stachyose can also be used as galactose donors, but give lower galactinol yields.

[0061] As used herein, the term "melibiose" or 6-O-α-D-galactopyranosyl-D-glucose is a reducing disaccharide of plant origin. Advantageously, melibiose can be isolated, for example, from Malvaceae, coffee seeds, or apples. Melibiose is commercially available in various forms, for example, as a powder. However, to date, melibiose is known to be very expensive. Methods for producing melibiose are known in the art.

[0062] The β-fructofuranosidase and / or α-galactosidase can be used in the incubation step as cell-free enzymes that do not require partial purification, as whole cell systems that have been physically or chemically pretreated to improve the permeability of the cell membrane (permeabilization) and improve mechanical stability, as encapsulated catalysts in which the free enzymes or whole cell systems are trapped, or immobilized on a carrier, e.g., a solid support (e.g., a chromatography resin).

[0063] Preferably, the β-fructofuranosidase and α-galactosidase, or both, are naturally expressed or recombinantly expressed, for example in a microorganism. For example, the native enzymes can be produced by fungi, such as the Aspergillus family, or yeasts, such as the Saccharomyces family. For example, the recombinant enzymes can be produced in expression hosts, such as Escherichia coli and Bacillus subtilis, or Pichia pastoris.

[0064] In one embodiment, the β-fructofuranosidase and / or α-galactosidase may be, for example, naturally occurring or genetically modified, e.g., resulting in increased activity of the β-fructofuranosidase and / or α-galactosidase. Preferably, the α-galactosidase may be in its naturally occurring form.

[0065] In a preferred embodiment, the fungus producing α-galactosidase can be, for example, Aspergillus niger, and the yeast producing β-fructofuranosidase can be, for example, Saccharomyces cerevisiae.

[0066] In another embodiment, the incubation pH of α-galactosidase is in the range of 3 to 9. In a preferred embodiment, the incubation pH is 3.5 to 8.5. In a more preferred embodiment, the incubation pH is 3.5 to 8.

[0067] In one embodiment, the incubation temperature for α-galactosidase may be comprised between 20 and 70°C, preferably between 25 and 65°C, more preferably between 30 and 60°C, even more preferably between 35 and 55°C, in particular between 40 and 50°C.

[0068] In one embodiment, β-fructofuranosidase and α-galactosidase are added, for example, separately or simultaneously, to a mixture of raffinose, or melibiose, and myo-inositol.

[0069] In a preferred embodiment, β-fructofuranosidase is added to raffinose, for example at 20-60° C., preferably at 25-55° C., in particular at 30-50° C., for 30-150 minutes, to allow the conversion of raffinose to melibiose and fructose.

[0070] In one embodiment, the incubation pH of the β-fructofuranosidase is in the range of 3 to 8. In a preferred embodiment, the incubation pH is 4 to 7.

[0071] In one embodiment, α-galactosidase can be added, for example, to melibiose in step a) where myo-inositol is added later, or to a mixture containing melibiose and myo-inositol. Preferably, myo-inositol can be added at the end of the reaction, for example in step a) where raffinose is quantitatively converted to melibiose and fructose. More preferably, myo-inositol can be added 30-150 minutes after the start of step A1). Advantageously, the enzymatic reaction can be stopped by inactivating the enzyme. For example, α-galactosidase can be stopped by increasing the temperature to about 65-80° C., preferably 72-78° C., for 1-10 minutes, preferably 3-6 minutes.

[0072] In a preferred embodiment, galactinol can be obtained by incubating a mixture containing myo-inositol and raffinose and / or stachyose with α-galactosidase at a pH between 3 and 9, preferably between 3.5 and 8.5, in particular between 3.5 and 8. In this case, it is not necessary to carry out step A1). Advantageously, both raffinose and stachyose can be used, since both act as galactose donors.

[0073] The reaction mixture containing galactinol obtained in step A1) or step A2) can be purified by purification methods known in the art, which may include separation by ultrafiltration membranes with a certain molecular weight cut-off value, or by various chromatographic methods, or by one or more of the following: liquid-solid separation, fermentation, crystallization and precipitation.

[0074] Thus, as a preferred purification step of the present invention, a fermentation process can be carried out, for example with a fungus, such as yeast (e.g. baker's yeast). Such purification by fermentation with fungi is known (McCready et al., Journal of the ASSBT: 127-132, 1965). Advantageously, since melibiose, galactinol and its isomers and myo-inositol are non-fermentable compounds, they are not affected by the yeast fermentation. In other words, they remain in the mixture obtained in step A1) and step A2). In contrast, since monosaccharides, such as glucose, fructose and galactose, are fermentable, yeast fermentation makes it possible to consume mainly these monosaccharides from the mixture.

[0075] In one embodiment, purification of the reaction mixture obtained in A1) and step A2) can be carried out by yeast fermentation. The yeast fermentation can be carried out for a time suitable to allow total consumption of the fermentable monosaccharides. Advantageously, the yeast fermentation can be carried out at 20-40° C., preferably at about 25-35° C., for 5-60 hours, preferably 10-50 hours, in particular 15-45 hours.

[0076] Additional purification steps may be carried out after the yeast fermentation described above.

[0077] In a preferred embodiment, the precipitation step with hydroalcoholic solvent can be carried out in the residual reaction mixture obtained after yeast fermentation. Advantageously, said residual reaction mixture can essentially contain galactinol, and also melibiose and myo-inositol. If this latter is present in the residual reaction mixture, it can be efficiently removed, at least partially or completely, for example by filtration after the precipitation step, followed by solvent removal, which can for example be done by solvent evaporation.

[0078] In a preferred embodiment, the residual reaction mixture can be decanted or preferably centrifuged after yeast fermentation. Solvent evaporation can be carried out on the supernatant at 40-80°C, preferably 50-75°C, more preferably 60-70°C, under reduced pressure. The resulting concentrated residual reaction mixture can be cooled at 15-0°C, preferably 2-5°C. Simultaneous cooling and addition of a hydroalcoholic solvent, preferably hydroethanol, improves the precipitation yield and thus the formation of crystals. At the end of this step, anhydrous crystals essentially composed by myo-inositol are obtained. These crystals can be washed with alcohol (e.g. ethanol) and filtered off. Advantageously, said myo-inositol crystals, which may be considered as a by-product, can be advantageously reused for another biocatalytic production of galactinol, thereby increasing the atom efficiency of such a subsequent process.

[0079] Thus, the hydroalcoholic solvent may be any solvent mixture containing at least one alcohol. Preferably, the hydroalcoholic solvent is mainly composed of one or more alcohols and water. Preferably, the hydroalcoholic solvent is composed of more than 20% by weight, at least 50% by weight, at least 70% by weight, at least 80% by weight, at least 90% by weight, at least 99% by weight of one or more alcohols and water, or (essentially) composed of one or more alcohols and water. As used herein, water may also include hot water or subcritical water. One or more aqueous buffers may also be used.

[0080] As used herein, the term "hydroalcoholic" may be understood in the broadest sense generally understood in the art. It may be any composition containing alcohol and water. The hydroalcoholic solvent may be any alcohol / water mixture. In a preferred embodiment, the solvent is an alcohol / water mixture containing 50-99% (v / v) alcohol, or containing 60-99% (v / v) alcohol, or containing 65-99% (v / v) alcohol, or containing 70-99% (v / v) alcohol, or containing 75-98% (v / v) alcohol, or containing 80-98% (v / v) alcohol, or containing about 97% (v / v) alcohol.

[0081] In a preferred embodiment, the alcohol may be selected from the group consisting of methanol, ethanol, propanol, butanol, pentanol, phenol, propanediol, glycerol, and mixtures of two or more thereof.

[0082] In a preferred embodiment, the hydroalcoholic solvent may be selected from mixtures of water / ethanol, water / methanol and water / propanol.

[0083] In a preferred embodiment, the hydroalcoholic solvent can be a hydroethanolic solvent. Thus, in a preferred embodiment, the solvent is an ethanol / water mixture containing 50-99% (v / v) ethanol, or containing 60-99% (v / v) ethanol, or containing 65-99% (v / v) ethanol, or containing 70-99% (v / v) ethanol, or containing 75-98% (v / v) ethanol, or containing 80-98% (v / v) ethanol, or containing about 97% (v / v) ethanol. In another preferred embodiment, the hydroalcoholic solvent can be a hydromethanolic solvent.

[0084] In one embodiment, galactinol can be purified from the reaction mixture after the above-mentioned precipitation step. Advantageously, for example, the purification step to obtain galactinol can be carried out by affinity chromatography, for example by activated carbon column. Such purification methods, which consist of absorbing the reaction mixture on a column followed by successive elution with a solvent, are known in the art.

[0085] In one embodiment of the invention, the reaction mixture can be absorbed onto the column and then eluted, for example, with a polar solvent. Suitable polar solvents are water and C 1 ~C 5 It may be a mixture with a primary alcohol having carbon atoms. The primary alcohol may be selected from methanol, ethanol, propanol, butanol and pentanol. Preferably, the alcohol may be selected from methanol and ethanol, in particular ethanol.

[0086] In one embodiment of the invention, the amount of alcohol in the hydroalcoholic solvent may be comprised between 1 and 20% (v / v) alcohol, preferably between 2 and 15% (v / v) alcohol, in particular between 3 and 10% (v / v) alcohol. The alcohol may be selected from methanol, ethanol, propanol, butanol and pentanol. Methanol and ethanol are preferred, and ethanol is most preferred.

[0087] In a preferred embodiment of the invention, elution with hydroalcoholic solvents can be carried out successively with increasing amounts of alcohol starting from 1%, preferably 2% (v / v) alcohol, in particular 3% (v / v) alcohol, and ending with 20% (v / v) alcohol, preferably 15% (v / v) alcohol, in particular 10% (v / v) alcohol. Thus, the elution steps can comprise two or more sequences, preferably three sequences.

[0088] In one embodiment of the invention, the last fractions eluted with 10-20% (v / v) alcohol can be combined and concentrated under reduced pressure to at least partially evaporate or completely evaporate the solvent to dryness to obtain a dry matter (physical form).

[0089] In a preferred embodiment of the present invention, the dried product can be solubilized in water and subsequently purified by affinity chromatography, such as with activated charcoal, as described above. This second purification can be performed identically to the first purification described above. Two fractions can be collected, one consisting of galactinol and the other galactinol isomers with α-linkages other than 3-α-, which can be selected from 1-O-α-D-, 2-O-α-D-, 4-O-α-D- or 5-O-α-D- or 6-O-α-D-galactopyranosyl-D-myo-inositol.

[0090] In a preferred embodiment of the present invention, both the galactinol and the galactinol isomer may have a purity of 60 to 100%, preferably 65 to 99%, more preferably 70 to 98%.

[0091] Optionally, after the aforementioned precipitation step, a decolorization step may be performed on the remaining mixture from which the myo-inositol crystals have essentially been removed. For example, decolorization may be performed using a hydrophobic resin.

[0092] Methods for the biocatalytic production of compounds are known in the art, so that the skilled person can select the best conditions for carrying out the method of the invention, i.e. in terms of temperature, enzyme concentration, pH, duration, solvent, etc., by referring to the enzyme information of the supplier.

[0093] The method according to the present invention can be carried out on an industrial scale.The industrial production of the galactinol of the present invention is carried out, for example, in a reactor known in the art.For example, the method can be carried out according to a batch process, a fed-batch process, a continuous process, or a combination thereof.The size of the reactor is not critical.

[0094] Therefore, the present invention also relates to compositions comprising 3-O-α-D-galactopyranosyl-D-myo-inositol obtainable (or obtained) from the biocatalytic process described above.

[0095] Another aspect of the present invention is a method for producing a composition comprising the steps of: a. 3-O-α-D-galactopyranosyl-D-myo-inositol as the active ingredient; and b. at least one additional cosmetically and / or pharma- ceutically acceptable ingredient other than 3-O-α-D-galactopyranosyl-D-myo-inositol; A cosmetic or pharmaceutical composition comprising: The cosmetic or pharmaceutical composition is a composition for topical use selected from the group consisting of a solution, a suspension, an emulsion, a cream, a paste, a gel, a lotion, a powder, a soap, a water containing a surfactant, an oil, a shampoo and a spray; and / or is a nutraceutical composition administered orally.

[0096] It will be understood that the definitions and preferred embodiments made in the context of galactinol according to the present invention apply mutatis mutandis to compositions containing it.

[0097] Here, 3-O-α-D-galactopyranosyl-D-myo-inositol may be considered as an active ingredient (or even as the only active ingredient). The term "active ingredient" may be understood in the broadest sense as an ingredient that can exhibit the desired intended activity, either alone or together with one or more other ingredients, such as one or more carriers that are inactive themselves or other active ingredients that may optionally act synergistically. Preferably, the active ingredient of the present invention is a cosmetically active ingredient.

[0098] The cosmetic or pharmaceutical composition can be used for any purpose and in any form.In a preferred embodiment, the composition is a composition for external use selected from the group consisting of a solution, a suspension, an emulsion, a cream, a paste, a gel, a lotion, a powder, a soap, a surfactant-containing water, an oil, a shampoo and a spray, or the composition is a nutraceutical composition for oral administration.

[0099] The composition may be or may be contained in a product selected from the group consisting of an emulsion, a gel, an ointment, a tonic, a liquid soap, a bar soap, a bath oil, a shower oil, a massage oil, makeup, a scalp treatment, an aftershave, a shaving product, a deodorant, a shower gel, a shampoo, and combinations of two or more thereof.

[0100] Optionally, the cosmetic or pharmaceutical composition may be a nutraceutical composition that can be orally administered to a subject. Optionally, the cosmetic or pharmaceutical composition may be included in a food, for example, a food supplement. Typically, the composition may have a systemic effect. The nutraceutical composition according to the present invention may be formulated together with an acceptable carrier into any form suitable for oral administration, for example, into a tablet, capsule, granule, powder, solution, emulsion or suspension.

[0101] In one embodiment, the composition may contain less than 20% by weight of galactinol. In a preferred embodiment, the composition contains 0.0001 to 15% by weight of galactinol, more preferably 0.001 to 10% by weight, even more preferably 0.005 to 8% by weight, especially 0.01 to 5% by weight of galactinol.

[0102] The at least one further cosmetically and / or pharma- ceutically acceptable ingredient other than galactinol can be any cosmetically and / or pharma- ceutically acceptable ingredient. In a preferred embodiment, the cosmetically and / or pharma- ceutically acceptable ingredient is or comprises at least one cosmetically and / or pharma- ceutically acceptable carrier.

[0103] As used herein, the terms "pharmaceutical acceptable carrier", "pharmaceutical acceptable excipient", "cosmetically acceptable carrier", "cosmetically acceptable excipient", "carrier" and "excipient" may be understood interchangeably in the broadest sense as any substance capable of supporting the cosmetic and / or pharmacological acceptability or usability of the compositions of the present invention containing galactinol.

[0104] The ready-to-use composition can preferably be a liquid formulation, particularly a composition suitable for topical administration. The storage form of said composition can also be liquid, but can also be a dry form (e.g., a powder, such as a powder that contains or consists of galactinol), or a paste or syrup, etc. Optionally, the dry form, paste or syrup can be dissolved or emulsified before being administered to the skin of the subject of interest.

[0105] The cosmetically and / or pharma- ceutically acceptable carrier may typically be selected from the list consisting of an aqueous buffer, saline, water, alcohol, vegetable oil, mineral oil, a polymer, or a combination of two or more thereof.

[0106] Optionally, the cosmetically or pharma- ceutically acceptable carrier may include one or more cosmetically or pharma- ceutically acceptable additives. In a preferred embodiment, such cosmetically and / or pharma- ceutically acceptable additives may be selected from the group consisting of fragrances / fragrances, dyes, pigments, emulsifiers, lubricants, chelating agents, acidity regulators, antimicrobial agents, preservatives, antioxidants, and combinations of two or more thereof. For example, the cosmetically and / or pharma- ceutically acceptable carrier may optionally include one or more detergents, triethanolamine, one or more fragrances, one or more foaming agents (e.g., sodium lauryl sulfate (SLS), sodium dodecyl sulfate (SDS), ... sulphate), one or more colorants (e.g., food colorants, pigments), one or more vitamins, one or more salts (e.g., sodium, potassium, calcium, zinc salts), one or more humectants (e.g., sorbitol, glycerol, butylene glycol, propylene glycol, mannitol, propylene glycol, polydextrose), one or more enzymes, one or more preservatives (e.g., benzoic acid, methylparaben), one or more antioxidants, one or more herbal and plant extracts, one or more stabilizers, one or more chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA), one or more polymers (e.g., carboxyvinyl polymers, carboxy The compositions of the present invention may contain, in addition to 3-O-α-D-galactopyranosyl-D-myo-inositol, one or more other active ingredients that act synergistically and / or complementarily with said 3-O-α-D-galactopyranosyl-D-myo-inositol without affecting its activity or its intended use as described above.

[0107] The composition according to the invention may be a cosmetic or may be included in a cosmetic. Harmful effects on the skin can be reduced or avoided following safety tests. Any route of administration that results in the desired purpose as claimed is suitable. Administration may be local or systemic. Preferably, administration is local. Administration may be topical, transdermal, oral, by injection (e.g., intravenous (iv), intraarterial (ia), intraperitoneal (ip), intramuscular (im), and subcutaneous (sc) injection), or intranasal. For example, the composition according to the invention may be formulated with an acceptable carrier in any suitable form for topical, oral, rectal, transmucosal, nasal, enteral, enteral and parenteral administration.

[0108] In a preferred embodiment, the composition is a composition for topical use.In a preferred embodiment, the composition is a composition for topical use, which is topically administered to a part of the skin, such as the face, scalp (including hair), or a part of the body.The skin can be any type of skin, including damaged skin, dry skin, healthy skin, normal skin, and oily skin.

[0109] As stated above, the galactinol of the present invention can be used for any purpose, preferably for cosmetic and / or pharmaceutical purposes.

[0110] A further aspect of the invention therefore relates to the cosmetic use of a composition comprising (or consisting of) 3-O-α-D-galactopyranosyl-D-myo-inositol as defined above for maintaining and / or restoring healthy skin through stimulation of its own endogenous defense mechanisms, for inducing the production of extracellular matrix components in skin cells, for stimulating growth factor production, for maintaining and / or strengthening the skin microbiota and more generally for improving the overall skin homeostasis and the overall skin appearance.

[0111] Surprisingly, the composition of the present invention makes it possible to improve the hydration of the skin, the appearance of the skin, stimulate the production of ECM components, induce the endogenous antioxidant defense mechanisms of the cells, and activate the endogenous cellular mechanisms involved in both the protection and repair of DNA and proteins. Thus, by achieving these functions, it is possible to improve and protect the overall appearance of the skin.

[0112] In a preferred embodiment, the endogenous defense mechanisms may be selected from antioxidant mechanisms, protection of the proteome, DNA repair enzymes, production of growth factors, or two or more of these.

[0113] In a preferred embodiment, the composition of the invention is for use in: a. Detoxifying skin cells through stimulation of antioxidant defense mechanisms; b. Protects skin cell proteome and skin barrier function through stimulation of repair proteins, c.To protect the DNA of skin cells through the stimulation of DNA repair genes, d. To protect skin cells from exposure to external stressors selected from UV radiation and environmental stressors; e. To promote skin firmness and elasticity through stimulation of extracellular matrix genes and growth factors; f. Promotes skin hydration through the production of hyaluronic acid and proteoglycans; g. To improve the micro-relief of the skin, h. To reduce skin wrinkles, i. A combination of two or more of these.

[0114] In a preferred embodiment, the use is for: - To improve the antioxidant and detoxifying properties of the skin microbiota; - To improve the metabolism of the host and the microbiota, - To prevent skin inflammation by modulating immune response elements, - to inhibit the pathogenicity of fungi by down-regulating their filamentous conversion, - To improve proteostasis in the host and skin microbiota.

[0115] Thus, improving proteostasis in the host and / or skin microbiota may include one or more of the following functions: - improving ribosome assembly; - improving protein transport; - improving polypeptide degradation; - Improving protein ubiquitination and subsequent degradation; - The synthesis process of proteins and amino acids, - improving chaperonin and folding processes; - Improve the regulation of glycoprotein synthesis and quality; - Reducing endoplasmic reticulum stress.

[0116] In one embodiment, the use is for: - improving the skin barrier function; - To strengthen the organization of the extracellular matrix and its structure.

[0117] As used herein, the term "metaproteomics / metaproteomics" can be understood as the proteomic analysis of a mixture of species, searching with a compilation of protein sequence databases representing at least some of the species in the mixture. The mixture may contain more than microbial species colonizing a mammalian host organism, for example, it may include host proteins. Indeed, metaproteomics allows access to functions activated at the human, bacterial and fungal level, whereas metagenomics does not. As used herein, "host" refers to the human body, preferably the skin.

[0118] As used herein, "proteostasis" can be understood as the dynamic regulation of a balanced functional proteome. The proteostasis network includes the competing integrated biological pathways within a cell that control the biosynthesis, folding, trafficking and degradation of proteins present within and outside the cell.

[0119] In a preferred embodiment, the use of the compositions of the present invention is for improving skin structure, delaying and / or preventing the appearance of wrinkles, improving skin hydration, treating loss of mechanical properties in the skin, in particular improving skin firmness, smoothness, thickness, tone, glow, texture and elasticity, maintaining and / or restoring skin barrier function, treating and / or preventing skin imperfections, treating and / or preventing skin damage, preventing or treating hair whitening, hair loss, promoting hair growth, hair density, or two or more of these.

[0120] Advantageously, it has been observed that the compositions of the invention make it possible to improve the overall appearance of the skin by acting at various levels, for example at the extracellular matrix level, at the protein and DNA level, by ensuring their protection.

[0121] As used herein, the term "overall appearance" of the skin and its appendages may be understood in the broadest sense as the overall condition (morphological and physiological) of the skin.

[0122] As used herein, the term "healthy skin" may be understood in the broadest sense as the ability of the skin to protect and repair itself to avoid excessive inflammation, abnormal pigmentation, poor barrier function and degradation of extracellular matrix components.

[0123] As used herein, the term "damage" can be understood in the broadest sense as any disturbance or malfunction in skin and its cells, especially in (otherwise) healthy skin.As used herein, damage can be induced by internal attack (aging, one or more genetic factors, one or more hormones) and external stress factors, such as light exposure (e.g., exposure to ultraviolet (UV) light, exposure to visible light and / or exposure to infrared light), exposure to certain pollution, exposure to one or more pathogens and / or allergens (including natural and chemical products), exposure to environmental stress factors (variable temperature: elevated temperature and humidity), and / or mechanical restraint (including those caused by daily habits, such as shaving, wearing gloves and masks).

[0124] As used herein, the term "annexe" may be understood in its broadest sense as it may include hairs and hair follicles.

[0125] Galactinol or a composition comprising it according to any of the embodiments of the present invention may have a local and / or systemic effect. In a preferred embodiment, when administered locally and / or topically, it has a (mainly or entirely) local effect.

[0126] In a preferred embodiment, said use is for the care of the skin, preferably said use is for improving the general appearance of any type of skin, including the scalp.

[0127] Stimulating the expression of ECM genes can be achieved by acting on their different components, in other words, the compositions of the invention make it possible to promote and / or stimulate the synthesis of ECM components.

[0128] Preferably, the stimulation of ECM genes can result in the promotion of the synthesis of proteins selected from collagen, procollagen, elastin, fibrin, fibrillin, fibronectin and laminin or mixtures thereof.The macromolecule can be a proteoglycan selected from decorin, perlecan, lumican, syndecan and versican or mixtures thereof; and / or non-sulfated glycosaminoglycan (e.g., hyaluronic acid).For example, decorin is the most highly expressed proteoglycan on human skin, and its size and content are known to decrease with aging.In one embodiment, stimulating the synthesis of hyaluronic acid can be achieved by promoting the expression of hyaluronic acid synthase 2 (HAS2).

[0129] Advantageously, since the ECM is targeted by acting on one or more of its components, the composition of the present invention allows to improve the mechanobiology of the skin in its entirety. Indeed, improving the mechanobiology of the skin can lead to the prevention of the appearance of fine lines and wrinkles, improvement of smoothness, firmness, tone, elasticity, thickness, texture and its elasticity, improvement of the skin barrier function. It is agreed that increasing and / or stimulating the production of hyaluronic acid and proteoglycans (e.g. LUM, SDC, VCAN) leads to an improvement of skin hydration. Moreover, these latter play an important role in participating in the mechanical strength of the skin. Moreover, since the production of ECM components (e.g. COL, FBN, FN, ELN and FBLN) is increased by the composition of the present invention, this can lead to an improvement of the mechanical strength of the skin.

[0130] Stimulating the expression of antioxidant defense genes can be achieved by promoting the expression of enzymes selected from superoxide dismutase 1 (SOD1), NAD(H) dehydrogenase quinone 1 (NQO1), heme oxygenase 1 (HMOX1), glutathione peroxidase 1 (GPX1), glucose-6-phosphate dehydrogenase (G6PD), or two or more. Thus, the composition of the present invention can be used for antioxidant and / or anti-free radical treatment.

[0131] Consequently, by acting on the ECM and antioxidant level, the compositions of the invention make it possible to delay and / or prevent the signs of skin ageing and to obtain healthy and younger skin.

[0132] Stimulating the expression of protein repair genes can be achieved by promoting the synthesis of transforming TGFβ1, nerve growth factor receptor (NGFR), NGF, HSPA1A, HSP90AA, HSP27, or two or more thereof. Thus, the composition of the present invention can be used to protect the skin from attacks caused by environmental stressors, such as heat, humidity, particulates, radiation (e.g., UV, infrared). Furthermore, promoting the production of HSP27 is involved in the final keratinocyte differentiation step and thus in improving skin barrier function. As a result, targeting these proteins can lead to improved skin homeostasis, particularly skin hydration, and protection of epidermal integrity.

[0133] Stimulating the expression of DNA repair genes can be achieved by promoting the synthesis of PCNA, MKi-67, GADD45A, or two or more of them.Targeting these genes can maintain the genetic integrity of cells and thus cell function.Therefore, the composition of the present invention can be used to protect and / or limit the aging, aging process and apoptosis of skin cells.

[0134] It will be understood that the definitions and preferred embodiments made in the context of a composition comprising (or consisting of) galactinol according to the invention apply mutatis mutandis to its (cosmetic) uses.

[0135] The present invention also relates to 3-O-α-D-galactopyranosyl-D-myo-inositol as defined above for use as a medicament, or to a cosmetic or pharmaceutical composition as defined above.

[0136] The present invention also relates to a composition comprising 3-O-α-D-galactopyranosyl-D-myo-inositol as an active ingredient for use in a non-therapeutic method for maintaining and / or improving the overall appearance of the skin and its annexes, said composition being for maintaining or improving skin homeostasis, for maintaining healthy skin, for improving skin firmness and / or elasticity, for protecting the skin against external stressors, for improving skin microrelief, for improving skin hydration and skin barrier function, for preventing and / or delaying the signs of skin ageing, and in respect of already aged skin, said composition being for protecting and enhancing the mobilization of its microbiota sources.

[0137] Throughout this specification and the claims, unless the context requires otherwise, open-ended words such as "comprise," "contain," "include," etc., and variations such as "comprises," "contains," "includes," "comprising," "containing," "including," etc., may be understood to mean the inclusion of a stated component, ingredient, integer or step, or group of ingredients, ingredients, integers or steps, but not the exclusion of any other components.

[0138] The terms "a" and "an" and "the" and similar references used in the context of describing the present invention (particularly in the context of the claims) should be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by the context. The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each separate value within that range. Unless otherwise indicated herein, each separate value is incorporated herein as if it were recited herein individually as well as in combination with each other. All method and procedure steps (including product-by-process steps) described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. The use of any and all examples or exemplary phrases (e.g., "such as," "for example") provided herein is intended merely to facilitate a better understanding of the invention and does not limit the scope of the invention as otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0139] It will be understood that the above aspects and embodiments can be combined in any manner and in any number to form additional embodiments. Any permutations and combinations of all elements described in this application should be considered to be disclosed by the description of this application, unless the context dictates otherwise.

[0140] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The methods and materials described herein are preferred, although other methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention.

[0141] All documents cited or referenced herein are incorporated by reference and may be used in the practice of the present invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

[0142] The following examples and figures are intended to illustrate further embodiments of the present invention without limiting its scope. EXAMPLES

[0143] example 1. Preparation of 3-O-α-D-galactopyranosyl-D-myo-inositol Hereinafter, 3-O-α-D-galactopyranosyl-D-myo-inositol will be referred to as galactinol.

[0144] In a 1 L reactor, 148 g of raffinose pentahydrate was dissolved at 45 °C and stirred at 300 rpm in 660 mL of 100 mM sodium phosphate buffer (pH 5.0 ± 1). 8 mL of β-fructofuranosidase (Invertin by Merck) was added. 商標 ; 2.4 KU / mL) was added and the reaction mixture was stirred for 2 h at 45° C. High performance liquid chromatography (HPLC) analysis showed complete conversion of raffinose to melibiose after 1 h.

[0145] 180 g of myo-inositol was added, and the pH was adjusted to 7.0±1 by adding sodium hydroxide. After the myo-inositol was completely solubilized, 1.85 g of α-galactosidase (Sumizyme 商標 AGS (30 KU / g) was added and the reaction was stirred at 45 °C while samples were taken every hour. After 5 h, HPLC analysis showed a galactinol concentration greater than 50 mM, and the reaction was quenched by heating the reaction mixture at 75 °C for 3-10 min.

[0146] After cooling to 30°C, 300 mg of MgCl2·6H20 and 25 g of freeze-dried baker's yeast were added and the resulting mixture was stirred at 30°C for 40 h. The reaction mixture was centrifuged and the supernatant was concentrated under reduced pressure at 70°C in a rotary evaporator. To induce crystallization, the concentrated solution was stirred at 70°C for 1 h. The temperature of the suspension was allowed to reach room temperature over 4 h, while aqueous ethanol was added as an antisolvent over the same time. After stirring overnight at room temperature, the suspension was stirred at 0°C for 2 h, after which the suspension was filtered. The crystals were washed with cold aqueous ethanol and both the filtrate and the washings were analyzed by HPLC. The filtrate and washings were combined and concentrated until the ethanol content was less than 2500 ppm, and the resulting solution was transferred to a hydrophobic resin (Dowex 商標 The mixture was passed through Optipore L-490.

[0147] The decolorized solution was analyzed by HPLC and water and glycerol were added to give an 80 / 20 glycerol / water (v / v) solution containing galactinol.

[0148] 1 shows the chemical structure of 3-O-α-D-galactopyranosyl-D-myo-inositol (galactinol) of the present invention, which is composed of a myo-inositol moiety and a galactose moiety.

[0149] HPLC chromatograms confirmed that the compositions of the present invention contained galactinol (data not shown). To perform this analysis, samples were analyzed by HPLC with an ELSD detector using a Luna Omeag Sugar column (250x4.6mm 3μm) at 1mL / min, 30°C under isocratic conditions (75% acetonitrile; 25% water). Furthermore, 1H and 13C NMR analysis confirmed that the compound eluting at a retention time (RT) of 25.5 minutes corresponds to 3-O-α-D-galactopyranosyl-D-myo-inositol, and the compound at RT of 26.5 minutes is an isomer of galactinol (α-OD bond).

[0150] 2. In vitro results: 2.1 Transcriptome research: In the following study, the ability of the compositions of the present invention to improve skin hydration, skin appearance, stimulate the production of ECM components, induce endogenous antioxidant defense mechanisms of cells, and induce endogenous cellular mechanisms involved in both DNA and protein protection and repair was demonstrated in normal human dermal fibroblasts (NHDF) using RT-qPCR technology (Taqman).

[0151] Hereinafter, galactinol and compositions containing it according to embodiments of the present invention are referred to as Gal and C-Gal, respectively.

[0152] 2.1.1 Method: Twenty-four hours after NHDF treatment with both Gal and C-Gal, mRNA expression was quantified.

[0153] The mRNA extraction was performed according to the supplier's recommendations (Qiagen). Briefly, lysis buffer RLT-Plus was added onto the cell pellet and the homogenous lysate was transferred onto a column. The eluate was mixed with one volume of 70° ethanol and transferred onto a pink "RNeasy" column. The column was washed with buffer. Elution was performed with RNase-free water and RNA quantification was performed with the "Nucleic Acid Quantification" software application by DO 260nm, 280nm, 320nm measurement (Biotek, Cytation3 & Take3 Plate). The quality of the mRNA was checked with a fragment analyzer (Advanced Analytical). Then, mRNA reverse transcription, which allows to obtain a cDNA template, was performed in a thermal cycler (Bio-Rad). qPCR was performed using the cDNA template. For the transcriptome study, Biorad's predesigned plates for ECM components, antioxidant genes and skin homeostasis were used. The data generated then allowed for automated calculation of mRNA expression fold changes and p-values ​​compared to untreated conditions using Bio-Rad CFX manager.

[0154] All data are statistically significant compared to the control condition (p-value <0.05).

[0155] 2.1.2 Results: 2.1.2.1 Stimulation of major skin ECM components FIG. 2 demonstrates the modulation of ECM gene and protein expression in NHDF treated with both Gal and C-Gal. Many ECM components, such as COL5A1, ELN, SDC4, VCAN, LAMC2, LUM and integrin alpha-1 (ITGA1), are known to confer tensile and mechanical strength, elasticity, resilience and hydration. These experimental results demonstrate that ECM components are targeted by Gal and C-Gal and confirm their ability to improve skin mechanobiology overall. Thus, both Gal and C-Gal can be used in preventing the appearance of fine lines and wrinkles, improving skin smoothness, firmness, thickness, tone, elasticity, texture and elasticity, and improving skin hydration. In other words, the composition of the present invention can be used as an active ingredient to improve the overall skin appearance and condition.

[0156] 2.1.2.2 Stimulation of the skin's endogenous antioxidant defense mechanisms As shown in Figure 3, antioxidant genes in NHDF were modulated by treatment with Gal and C-Gal. Briefly, NQO1 is a detoxification enzyme via the quinone / hydroquinone system. HMOX1 is involved in cellular defense against oxidative stress, and GPX1 is involved in the detoxification of hydrogen peroxide (H 2 O 2 Known as one of the most important antioxidant enzymes in humans in detoxifying the body's metabolic pathway, G6PD plays a role in providing energy to cells by maintaining levels of NADPH, which in turn maintains the supply of glutathione in cells, which is used to scavenge free radicals that cause oxidative stress.

[0157] 2.1.2.3 Stimulation of genes involved in the overall maintenance of skin homeostasis, particularly growth factors, heat shock proteins, DNA repair and cell proliferation markers As shown in Figure 4, in NHDF, among the modulated genes, upregulation of growth factors such as TGFβ1 and NGF was demonstrated. TGFβ1 is involved in the control of cell growth, cell proliferation, cell differentiation and apoptosis. NGF is involved in the cell proliferation cycle. In addition, upregulation of heat shock proteins, mainly HSPA1A and HSP90AA, was observed. HSPA1 is known to promote proper folding of new and misfolded proteins to stabilize and degrade modified proteins. HSP90AA promotes maturation, structural maintenance and proper regulation of proteins involved in cycle control, for example. And upregulation of mRNA expression of PCNA, MKi67 and GADD45A by Gal and C-Gal was found. These genes code for DNA repair and cell proliferation proteins. PCNA acts as a processivity factor for DNA polymerase delta and is essential for replication. MKi-67 is a marker of cell proliferation. The GADD45A gene is a member of a group of genes whose transcript levels are increased under stressful growth arrest conditions and following treatment with DNA damaging agents.

[0158] The results of these experiments demonstrated the ability of both Gal and C-Gal to: improving cell proliferation, limiting and / or preventing cellular senescence and therefore more generally preventing and / or slowing down the signs of skin aging, - improves the skin's homeostasis and therefore protects it from various stresses that contribute to alterations in the skin's homeostasis; - Protect proteins (e.g. enzymes, nuclear factors) from alterations, - Repairing oxidative damage to DNA (e.g. due to external aggressions such as UV rays, microparticles, etc.), - Improve skin remodelling and, more generally, the overall appearance of the skin and its condition consistent with the results obtained with the ECM detailed above.

[0159] As a result, the compositions of the present invention may be used to prevent the appearance of fine lines and wrinkles, improve skin smoothness, prevent hair loss and improve hair density.

[0160] 2.2 Evaluation of skin cytoprotective properties using cell morphology observation In the following studies, the ability of Gal and C-Gal to protect skin cells (NHEK and NHDF) from various stress factors, such as UV radiation (UVB and UVA) or environmental factors (humidity and temperature) was evaluated.

[0161] 2.2.1 Method: Briefly, a bright-field imaging microscope was used to study the effect of UVB irradiation on the morphology of NHEKs in various experimental conditions in the presence or absence of Gal and C-Gal: - "Preventive" condition: Gal or C-Gal was administered with UVB irradiation (30 mJ / cm 2 ) was applied to NHEK for 24 hours before and after 24 hours, the cell morphology was observed. - "Therapeutic" condition: Gal or C-Gal was applied after UVB irradiation and the morphology of the cells was observed after 24 hours. - Combined condition "prophylactic and therapeutic": Gal or C-Gal was applied before and after UVB irradiation and the morphology of the cells was observed after 24 hours.

[0162] The same experiment as above was repeated using UVA irradiation on NHDF under the following conditions: - "Therapeutic" condition: Gal or C-Gal was administered with UVA irradiation (10 J / cm 2 ) and the cell morphology was observed 24 hours later.

[0163] Finally, the effect of environmental stress on cell morphology was examined (using bright-field imaging microscopy) in NHEKs treated with or without Gal or C-Gal. The environmental stresses applied were heat stress (temperature increase from 37 to 44°C for 30 min), or moisture stress (change in relative humidity from 90% to 45%), or a combination of both stresses (heat + moisture stress).

[0164] 2.2.2 Results (data not shown) The harmful effects of UVB, UVA and environmental stressors (heat and humidity) were observed in NHEK and NHDF cells. Indeed, during stress conditions, in UVA and UVB conditions, fewer cells were present (suggesting cell death under these stress conditions) and the morphology of the cells was clearly altered. Indeed, healthy keratinocytes have a polygonal shape, whereas during stress conditions they appear more elongated (like fibroblasts). In contrast, in environmental stress conditions, a decrease in cell number was observed. In parallel, strong changes in cell morphology appeared, suggesting a change in their homeostasis. Thus, the composition of the present invention is suitable for protecting cells against UVB, UVA and environmental damage. Indeed, more cells are present in stress-treated conditions with Gal and C-Gal compared to stress conditions alone. Moreover, the normal expected cell morphology was observed in the treated groups.

[0165] 2.3 Evaluation of the skin cytoprotective properties of the composition via stimulation of HSP27 protein expression 2.3.1 Method: NHDFs were treated with Gal or C-Gal for 24 h (treated cells). Cells were then fixed for 30 min and immunostained with primary antibody HSP27 (ab62339, Abcam) and exposed to secondary antibody conjugated with Alexa fluor 568 (A1101, Thermofisher). Images were obtained with a Nikon microscope (Eclipse Ti2). Quantification of HSP27 was measured by image analysis of fluorescence intensity per cell using Image J software. Statistical analysis (Dunnett) was performed with Minitab software (*p<0.05 compared to control). Results were obtained by performing three independent assays.

[0166] 2.3.2 Results In Figure 5, a clear stimulation of HSP27 expression in treated cells was observed under basal conditions (i.e., non-stress conditions). This result suggested that Gal or C-Gal may have preventive and protective effects on skin cells.

[0167] 2.4 Evaluation of skin hydration properties To examine whether the composition of the present invention can improve skin hydration, its effect on hyaluronic acid (HA) production by NHEK cells was evaluated.

[0168] 2.4.1 Method: NHEK were cultured in DERMALIFE medium 商標 K and penicillin / streptomycin in 5% CO in air 2 The cells were incubated for 24 hours at 37°C in a humidified atmosphere containing

[0169] After 24 hours, the cells were incubated with 10 μL of C-Gal diluted in 90 μL of culture medium per well, or with a final 10 μL of C-Gal as a positive control. -8 M EGF (Gibco 商標 , ref: 2116096) and then incubated at 37 °C for 12 h at 5% CO in air. 2The cells were cultured for 48 hours at 37 °C in a humidified atmosphere containing 100 mM NaCl. The supernatant was then collected and subsequently centrifuged to quantify the hyaluronic acid produced by the cells. To standardize each condition in which hyaluronic acid was quantified, the total protein content was quantified using the Bradford method. Finally, the quantification of hyaluronic acid was performed by ELISA technique (Hyaluronan DHYALO kit from R&D System) according to the supplier's recommendations.

[0170] All data were expressed as mean ± standard deviation. Statistical significance of the data was determined by two-way analysis of variance using Minitab software (*p<0.05 compared to control). Results were obtained by performing three independent assays.

[0171] 2.4.2 Results Figure 6 shows the ability of C-Gal to promote the synthesis of HA by NHEKs. As a result, skin hydration is improved by increasing the production of HA in skin cells. Furthermore, RT-QPCR analysis showed that C-Gal significantly stimulated the mRNA expression of HAS2 (hyaluronan synthase 2, fold change x2.2), an enzyme involved in HA synthesis.

[0172] 3. Clinical Trials: 3.1 Evaluation of the Properties of the Compositions of the Invention to Improve the Biomechanical Properties of Human Skin 3.1.1 Method: A randomized, double-blind, placebo-controlled clinical trial was conducted. The study adhered to the principles of the Declaration of Helsinki and the International Conference on Harmonization Good Clinical Practice guidelines as applicable to non-pharmaceutical trials.

[0173] Twenty Caucasian women were enrolled in this clinical trial, which lasted for 28 days. The volunteers ranged in age from 50 to 60 years, exhibited wrinkles, and had dull, uneven features due to daily exposure to light. The subjects applied a placebo and a formulation containing 0.3% of the composition of the present invention to half of their face.

[0174] [Table 1] The parameters were measured by the AEVA-HE device (by EOTECH) and the cutometer (courage and Khazaka). The AEVA-HE device was positioned to take 2D and 3D high-resolution photographs of the wrinkle volume. The cutometer allows the determination of the Ue and Ur parameters, two parameters that measure skin elasticity and skin firmness, respectively.

[0175] 3.1.2 Results: Figure 7 (A and B) shows a significant visible improvement in the biomechanical properties of the skin after 28 days of use of the composition of the invention, compared to the starting date (D0). Indeed, after 28 days of application of the cream of Table 1, both parameters Ue (skin elasticity) and Ur (skin firmness) increase. These parameters are known to decrease significantly with chronological aging, but also during photoaging. It can therefore be considered that the composition of the invention can improve the elasticity, firmness and smoothness of the skin, and more generally the overall appearance of the skin.

[0176] All data were expressed as mean ± standard deviation. Statistical significance of the data was determined by two-way analysis of variance using Minitab software ( *** p<0.001 compared with D0; ** p < 0.01 compared with D0; * p<0.05 compared to D0). Results were obtained by performing three independent assays.

[0177] Furthermore, measurements of wrinkle volume before (representing D0) and after 28 days of treatment with the composition of the present invention showed a significant reduction in wrinkle volume in the treatment group (Figure 7C).This result demonstrates the ability of the composition of the present invention to significantly reduce wrinkles.

[0178] 4. Study of the metaproteomic effect of the compositions of the invention: 4.1 Materials and Methods: Twenty volunteers were recruited for metaproteomic analysis. Samples were taken before (day 0) and after (day 28) treatment with C-Gal on one side of the face and with placebo on the other side.

[0179] The aim of the metaproteomics study was to simultaneously investigate proteins from the host (the human body) and its microbiome (bacteria and fungi). HolXplore, developed by Phylogen SA (France), 商標 A bioinformatics tool called dsDNA was used for taxonomic and functional analyses. The analyses were performed according to the supplier's recommendations. A data processing module for host and microbiome outputs of omics comparative studies is obtained. This includes taxonomic analyses of diversity combined with different experimental conditions, bacterial, fungal and human taxa, parallel functional analyses on different functions and / or correlations between taxa.

[0180] Briefly, proteins, whatever their taxonomic origin, were extracted from skin swabs and digested. Peptide extracts were labeled and analyzed by MS / MS fragmentation and nanoLC HRAM MS techniques (nanochromatography coupled with high-resolution accurate mass spectrometry).

[0181] Proteins were then identified using the SEQUEST-HT algorithm against all bacterial, fungal and human reference proteomes obtained from UniProt KB, together with sequences of the most common experimental contaminants in LC-MS / MS, excluding sequences also found in the human reference proteome.

[0182] Taxonomic analysis focused on the microbiome composition in terms of taxon abundance, which allows the taxonomic assignment of each protein according to their peptide classification.

[0183] The identified taxa were selected in three different taxa: host (human), bacteria and fungi. Diversity analysis then highlighted the differences in microbiome composition and diversity between the sample groups. The abundance of each taxon was calculated as the sum of its associated protein abundances. Their abundances were used as input data to measure "α-diversity" and "β-diversity" using the "diversity" and "vegdist" functions, respectively, of the "vegan" R package: - Alpha diversity measures diversity within a sample - β diversity measures the diversity between samples and the separation of samples according to their microbiome composition.

[0184] Functional analysis focused on the function of the identified proteins. Each protein was functionally annotated and enrichment analysis was performed to highlight the altered biological processes. The majority of microbial proteins lack functional annotations. For this, protein sequences (recovered from Uniprot and Uniparc databases) were submitted to the bioinformatics known tool EggNOGmapper to associate each protein with its closest annotated orthologues. The functional annotations of the orthologues were used for further analysis.

[0185] 4.2 Results: 4.2.1 From the taxonomic analysis: The analysis of β-diversity did not highlight any separation of samples according to the two treatments (placebo vs. C-Gal). In contrast, the α-diversity analysis highlighted significant differences in sample diversity between the two treatments, showing a greater diversity with respect to the active group C-Gal, without perturbation of the microbiome composition. Thus, the composition of the invention is safe for the skin microbiota and can therefore be considered as "microbiota-friendly". Interestingly, thanks to the taxonomic analysis, an upregulation of bacteria of the genus Amycolatopsis, whose secondary metabolites are known to exert antimicrobial, anticancer and antioxidant activity, was observed.

[0186] 4.2.2 From the functional analysis: C-Gal was demonstrated to be modulated by: - Antioxidant and detoxification properties of skin microbiota treated with C-Gal: The abundance of proteins related to oxidative stress was modulated in fungi and bacteria. Peroxiredoxins are antioxidant enzymes that also control peroxide levels and mediate related signaling events (particularly in conjunction with thioredoxin). Three peroxiredoxins and two thioredoxins were regulated. Several proteins potentially involved in antioxidant responses were also regulated, such as SOD, an uncharacterized protein orthologue of GDH3 (Glycine cleavage system H protein-3) that plays a central role in maintaining redox balance, and luciferase protein involved in the oxidation of long-chain aldehydes. Finally, proteins involved in formaldehyde detoxification and other types of cellular stress, such as genotoxic stress from fungal origin, were also regulated.

[0187] - Host and microbiota metabolism: Energy metabolism allows cells to generate energy from various sources. Glucose is one of the main sources of energy. The results clearly highlighted the upregulation of glucose metabolism (mainly composed by GAPDH protein) and lipid metabolism in bacteria and fungi, as well as lipid metabolism (mainly related to perilipin-5 (PLIN5), a protein involved in lipid storage) in the host. C-Gal upregulated a series of proteins involved in cell wall components production such as peptidoglycan, lipopolysaccharides and mycolic acids.

[0188] - Anti-inflammatory properties: Immunity allows the host to control its microbiome to avoid infection and dysbiosis, but chronic inflammation can result in skin dysfunction and disease. With regard to immune components, host control proteins suggested a reduction in inflammation, mainly due to downregulation of complement C8β chain (C8B: a component of the membrane attack complex), and upregulation of negative regulators of inflammatory signaling (CD81, receptor tyrosine protein phosphatase epsilon (PTPRE), and Ras-related protein Rab-7b (RAB7B)). CD81 increases the breakpoint cluster region protein (BCR) signaling pathway, PTPRE inhibits cytokine production and degranulation, and RAB7B negatively regulates the Toll-like receptor (TLR) pathway and subsequent inflammation. Furthermore, C-Gal modulated cathelicidin antimicrobial peptides, which are direct agents against microbial populations.

[0189] - Inhibition of fungal pathogenicity: Microbiota proteins can also affect host functions. Some commensal fungi are able to switch from yeast to filamentous forms, a morphogenesis essential for their pathogenic potential. The filamentous forms can invade the keratinized epithelium, leading to the disruption of the epidermal barrier function and ultimately inducing disease. Proteins involved in filamentous growth were regulated, such as ELM1, ASC1 and orthologues of the transcription factor RBF1, which can inhibit filamentous growth. These results suggested a downregulation of the filamentous forms, leading to a reduced invasion of the epithelial barrier, ensuring the maintenance of better skin health.

[0190] - Protein regulatory pathways, also called "proteostasis": some of them are ribosomal proteins from bacterial or human origin. Proteins involved in protein trafficking in the host, such as peptidases involved in polypeptide degradation and enzymes involved in protein ubiquitination and subsequent degradation, were also regulated. In addition, proteins were folded in the endoplasmic reticulum. The accumulation of misfolded or unfolded proteins leads to endoplasmic reticulum stress (ER stress), which is closely related to oxidative stress. Chaperonins are essential for correct protein folding and some of them (mainly from bacterial origin) were regulated. Three peptidylproline isomerases (PPIases), facilitators of this folding process, were upregulated. The downregulation of reticulocalbin-1 supported the hypothesis that ER stress is reduced due to its role in ER stress-induced apoptosis.

[0191] - C-Gal showed an improvement in skin barrier function: Besides inflammation and fungal invasion, various parameters can affect the skin barrier integrity of the host cells, such as cell-cell adhesion and cell-matrix adhesion. p53 apoptosis effector associated with PMP-22 (PERP) is a component of the desmosome, and increased desmosome assembly is an indicator of a stronger epithelial barrier and skin integrity. Finally, the precursors of the cornified envelope of the stratum corneum were also regulated.

[0192] - Enhanced ECM organization and structure: Matrilysin is an enzyme capable of degrading ECM, and its downregulation may be associated with stronger ECM structure or reduced tissue remodeling. Furthermore, C-Gal stimulated glycosaminoglycan (GAG) synthesis via upregulation of UDP-glucuronic acid decarboxylase 1, a key enzyme involved in GAG synthesis.

[0193] - The experimental metaproteomic results obtained with the composition of the invention, combined with the above experiments, confirm the ability of said composition to improve the overall appearance of the skin, for example to make it healthier through the preservation of its microbiota sources and the enhanced mobilization of said sources. The composition of the invention may therefore be used to obtain both "pre-ageing" aging and "pro-aging" effects.

Claims

1. A composition comprising 3-O-α-D-galactopyranosyl-D-myo-inositol as an active ingredient.

2. 2. The composition of claim 1, further comprising a galactinol isomer having an α-linkage other than a 3-α-D linkage selected from 1-O-α-D-, 2-O-α-D-, 4-O-α-D-, 5-O-α-D-, or 6-O-α-D-galactopyranosyl-D-myo-inositol.

3. 3. The composition according to claim 1 or 2, wherein the molar ratio between 3-O-α-D-galactopyranosyl-D-myo-inositol in the composition and galactinol isomers having α-linkages other than 3-α-D-linkages is approximately from 3:1, preferably from 2:1, in particular from 1.5:

1.

4. 4. The composition according to claim 1, wherein the mixture comprising 3-O-α-D-galactopyranosyl-D-myo-inositol and galactinol isomers represents 20% by weight, preferably 25% by weight, in particular 30% by weight, relative to the total weight of the composition.

5. The composition according to any one of claims 1 to 4, further comprising melibiose or myo-inositol or a monosaccharide or a mixture thereof, wherein the monosaccharide is selected from galactose, glucose, fructose or a mixture thereof.

6. based on the total weight of the composition, 0-25% by weight of melibiose, 0-40% by weight of myo-inositol, - 0-30% by weight of monosaccharides The composition according to any one of claims 1 to 5, comprising:

7. 1. A biocatalytic process for the preparation of galactinol having the formula 3-O-α-D-galactopyranosyl-D-myo-inositol, comprising the steps of: A1) providing melibiose or incubating raffinose with β-fructofuranosidase at a pH between 3 and 8 to obtain melibiose; and incubating a mixture comprising melibiose and an acceptor selected from pinitol and / or inositol isomers including myo-, chiro-, scyllo-, chiro-, neo-, allo-, epi- and cis-inositol, preferably myo-inositol, with α-galactosidase at a pH between 4 and 9; or A2) if step A1) was not performed, incubating a mixture comprising an acceptor selected from pinitol and / or inositol isomers including myo-, chiro-, scyllo-, chiro-, neo-, allo-, epi- and cis-inositol, preferably myo-inositol, and raffinose and / or stachyose with α-galactosidase at a pH between 3 and 9; B) Purifying 3-O-α-D-galactopyranosyl-D-myo-inositol from the mixture of compounds obtained in step A1) or step A2).

8. The process according to claim 7, wherein the β-fructofuranosidase is added to the raffinose at a temperature in the range of 20 to 60° C., preferably at 25 to 55° C., in particular at 30 to 50° C., for a time in the range of 15 to 160 minutes.

9. 9. The method according to claim 7 or 8, wherein myo-inositol is added after a time period between 30 and 150 minutes after step A1) is started.

10. 10. The method according to any one of claims 7 to 9, wherein purification of the reaction mixture obtained in step A1) or step A2) is carried out by yeast fermentation.

11. The method according to any one of claims 7 to 10, wherein a precipitation step with a hydroalcoholic solvent is further carried out on the residual reaction mixture obtained after yeast fermentation.

12. a. 3-O-α-D-galactopyranosyl-D-myo-inositol as the active ingredient; and b. at least one additional cosmetically and / or pharma- ceutically acceptable ingredient other than 3-O-α-D-galactopyranosyl-D-myo-inositol; A cosmetic or pharmaceutical composition comprising: The cosmetic or pharmaceutical composition is a composition for topical use selected from the group consisting of a solution, a suspension, an emulsion, a cream, a paste, a gel, a lotion, a powder, a soap, a surfactant-containing water, an oil, a shampoo and a spray; and / or is a nutraceutical composition administered orally.

13. Cosmetic use of a composition comprising or consisting of 3-O-α-D-galactopyranosyl-D-myo-inositol according to any one of claims 1 to 6 and 12 for maintaining and / or restoring healthy skin through stimulation of its own endogenous defense mechanisms, for inducing the production of extracellular matrix components in skin cells, for stimulating growth factor production, for maintaining and / or strengthening the skin microbiota and more generally for improving the overall skin homeostasis and the overall skin appearance.

14. 14. The cosmetic use according to claim 13, wherein the endogenous defense mechanisms are selected from antioxidant defense mechanisms, protection of the proteome, DNA repair enzymes, production of growth factors, or two or more thereof.

15. 15. The cosmetic use according to claim 13 or 14, wherein the use is for: a. To detoxify cells through stimulation of antioxidant defense mechanisms; b. To protect the skin cell proteome and skin barrier function through stimulation of repair proteins; c. To protect the DNA of skin cells through the stimulation of DNA repair genes; d. to protect skin cells from exposure to external stressors selected from UV radiation and environmental stressors; e. To promote skin firmness and elasticity through stimulation of extracellular matrix genes and growth factors; f. To promote skin hydration through the production of hyaluronic acid and proteoglycans; g. To improve the micro-relief of the skin, h. To reduce skin wrinkles; i. For any combination of two or more of these.

16. 14. The cosmetic use according to claim 13, wherein the use is for: - To improve the antioxidant and detoxifying properties of the skin microbiota; - To improve the metabolism of the host and the microbiota, - to prevent skin inflammation by modulating immune response elements, - to inhibit the pathogenicity of the fungus by down-regulating its filamentous transformation, - To improve proteostasis in the host and in the skin microbiota.

17. The cosmetic use according to any one of claims 13 to 16, wherein improving proteostasis in the host and / or skin microbiota comprises one or more of the following functions: - improving ribosome assembly; - improving protein transport; - improving polypeptide degradation; - improving protein ubiquitination and subsequent degradation; - improving the processes of protein and amino acid synthesis; - improving chaperonin and folding processes; - improving the control of glycoprotein synthesis and quality; - Reducing endoplasmic reticulum stress.

18. The cosmetic use according to any one of claims 13 to 17, wherein the use is for: - To improve the skin barrier function, - To strengthen the organization and structure of the extracellular matrix.

19. 19. Cosmetic use according to any one of claims 13 to 18, wherein said use is for improving skin structure, for delaying and / or preventing the appearance of wrinkles, for improving skin hydration, for treating loss of mechanical properties in the skin, in particular for improving skin firmness, smoothness, thickness, tone, lustre, texture and elasticity, for protecting and / or restoring the skin barrier function, for treating and / or preventing skin imperfections, for treating and / or preventing skin damage, for preventing or treating hair whitening, hair loss, for promoting hair growth and hair density, or for two or more of these.

20. 1. A composition comprising or consisting of 3-O-α-D-galactopyranosyl-D-myo-inositol as an active ingredient for use in a non-therapeutic method for maintaining and / or improving the overall appearance of the skin and its appendages, for maintaining or improving skin homeostasis, for maintaining healthy skin, for improving skin firmness and / or elasticity, for protecting the skin against external stressors, for improving skin relief, for improving skin hydration and skin barrier function, for preventing and / or delaying the signs of skin aging and, in respect of already aged skin, for protecting and enhancing the mobilization of its microbiota sources.

Images