Compositions comprising kombucha and their uses

Abstract

The invention is generally directed to identification and use of a kombucha extract, alpha-glucan oligosaccharide, or a mixture thereof that is capable of increasing skin barrier protection as assessed by ability to increase cell differentiation certain biomarkers of as measured by expression of filaggrin and involucrin in particular. A composition may be used in a method of improving an aesthetic appearance of skin of a human affected by weakened skin barrier comprising topically applying thereto a therapeutically effective amount of an active agent, wherein the active agent increases synthesis of involucrin and / or filaggrin by greater than 40% when applied ex vivo to human skin keratinocytes at a concentration of at least 0.45% w / w in a cosmetically acceptable vehicle.

Description

COMPOSITIONS COMPRISING KOMBUCHA AND THEIR USESCLAIM OF PRIORITY

[0001] This patent application claims the benefit of priority to U.S. Provisional Application Serial No. 63 / 500,428, filed May 5, 2023, and Netherlands Application Serial No. 2035681, filed August 25, 2023, which are incorporated by reference herein in their entireties.BACKGROUND

[0002] It is well established that a complex interplay of comeocytes and intercellular lipids in the stratum corneum of the skin is responsible for the skin barrier against environmental factors. A cornified cell envelope, which is composed of involucrin, loricrin, filaggrin, and other proteins, is a component of fully differentiated epidermal keratinocytes and corneocytes, and it is important in the skin barrier. (Lee et. al Ann Dermatol. 2014 Feb; 26(1): 134-137. Published online 2014 Feb 17. doi: 10.5021 / ad.2014.26.1.134).

[0003] The skin barrier, also referred to as the epidermal permeability barrier, protects against infection and poisoning, prevents desiccation and is essential for terrestrial life. Barrier function is conferred by the outer layer of epidermis, the stratum corneum which consists of dead, keratin-filled cells embedded in a lipid matrix. Stratum corneum is formed from granular layer keratinocytes during terminal differentiation of normal adult epidermis. A rapid aggregation of the keratin cytoskeleton, which causes a collapse of the granular cells into flattened a nuclear squames, is a key step in formation of the outermost barrier layer of the skin. This condensed cytoskeleton is cross linked by transglutaminases during formation of the cornified cell envelope (CE). Transglutaminases are expressed and activated during terminal differentiation of keratinocytes. The membrane-bound form of the transglutaminase- 1 forms ester bonds between specific glutaminyl residues of human involucrin during formation of the cornified cell envelope enzyme. The CE not only prevents water loss but also impedes the entry of allergens and infectious agents. During the last stage of its terminal differentiation of keratinocytes is the formation of a cross linked envelope. This envelope is made up of membrane and cytosolic proteins cross linked by glutamyl lysine isopeptide bonds.

[0004] Involucrin, being a keratinocyte protein which appears first in the cytoplasm and later becomes cross linked to membrane proteins by transglutaminase, is the most abundant component is of the envelope. It is a highly reactive, soluble, transglutaminase substrate proteinin keratinocytes of epidermis and other stratified squamous epithelia. It first appears in the cell cytosol, but ultimately becomes cross-linked to membrane proteins by transglutaminase thus helping in the formation of an insoluble envelope beneath the plasma membrane functioning as a glutamyl donor during assembly of the cornified envelope.

[0005] Filaggrin is a protein which is expressed in granules in the granular layer of interfollicular epidermis which is predominantly composed of the protein pro-filaggrin. Upon terminal differentiation of granular cells, pro-filaggrin is proteolytically cleaved into filaggrin peptides. Filaggrin aggregates the keratin cytoskeleton and is therefore a key protein in facilitating epidermal differentiation and maintaining barrier function. It is largely responsible for how well your skin retains moisture, and determines how hydrated and healthy your skin is. Filaggrin is essential for the production of Natural Moisturizing Factors (NMF) - substances that maintain skin hydration and skin barrier. People with eczema-prone skin may be deficient in the gene for making Filaggrin which may result in a shortage of this protein. A lack of Filaggrin makes it easier for moisture to escape as the skin barrier is compromised, making the skin vulnerable and prone to dehydration.

[0006] As described in EP1555999BA, Kombucha (INCI: Saccharomyces / Xylinum / Black Tea Ferment) is a fermented tea a tea which has undergone a microbiological transformation. This transformation is a fermentation by a symbiote of yeast of the genus Saccharomyces which nidifies within a polysaccharide matrix produced by a xylinum bacterium. The exact composition of this symbiose (in particular the proportions of each of the species) varies with the geographical and climatic conditions and depends on the wild local subspecies of yeast and bacteria; nevertheless, mention may be made, among others, without this list being limiting: Saccharomyces ludwigii, Saccharomyces apicalutus species, Bacterium xylinoides, Bacterium gluconicum, Schizosaccharomyces pombe, Acetobacter ketogenum, Torula species, Pichia fermentans and other yeasts. In the literature on kombucha, this symbiote of yeasts and bacteria is also called "fungus", "long-life winner" and many synonyms.

[0007] This fermentation is entirely original since the alcohol produced by the yeast from sugar is transformed by the bacterium into various acids, glucuronic, lactic, usnic and especially acetic, which gives its acidulated taste to kombucha with a final pH of between 2,5 and 4. On the other hand, the xylinum bacterium, termed "acetic bacterium", also uses the sugar present in tea and transforms the sucrose into cellulose microfibrils, thus constituting the support membrane in which the yeast nidifies and develops.

[0008] The products of yeast metabolism are excreted in kombucha and consist of numerous vitamins such as Bl, B2, B3 and B12, cofactors essential to the growth of bacteria.

[0009] Kombucha, also called "long-life mushroom tea", is a popular therapeutic remedy known for a long time by various names [FRANK G., 1999], It is a refreshing drink with a yellow-amber color with a sweet-acidic taste of cider. Its effect has been known and appreciated for generations by many people, especially in the countries of East Asia. The tea used for the manufacture of kombucha can be of any kind and of any origin, in particular Camellia sinensis varieties sinensis or assamica. All varieties of green tea, semi-fermented tea, black tea, smoked black tea, yellow tea, dark tea, white tea, herbal or fruit tea, infusion, can be used as a basis for the manufacture of kombucha. Preferably, all varieties of green tea, semifermented tea, black tea, smoked black tea, yellow tea, dark tea, white tea (Camellia sinsensis) and more particularly black tea.

[0010] Alpha-Glucan Oligosaccharide (INCI: Alpha-Glucan Oligosaccharide) is obtained by enzymatic synthesis from natural sugars (sucrose and maltose). This product is used for the protection and biostimulation of natural skin defenses. It is a bioselective substrate for beneficial microbial flora to the detriment of pathogens and undesirable flora. It acts of the skin’s immune system by stimulating the production of antimicrobial peptides, limiting the pro-inflammatory cascade and preserving the barrier function. It offers gentle protective and restoring action for all types of skin and the scalp.

[0011] Thermus thermophilus is a Gram-negative bacterium originally isolated from a thermal vent in Izu, Japan. It is used in a range of biotechnological applications because it is a source of enzymes, in particular thermozymes. Thermus thermophilus ferment (INCE Thermus Thermophillus Ferment (and) Glycerin)) has antioxidant, radical scavenging properties, is a source of alpha-galactosidase and helps maintain a firm feel of the skin.

[0012] Agents that enhance and improve the natural skin barrier function as described above are useful in cosmetic products, e.g. by enhancing the aesthetic appearance of skin by improving retention of moisture in the skin, lessening dry skin formation and generally keeping the skin in a healthy and visually appealing condition.

[0013] There remains a need for methods of identifying active ingredients that will prove effective at improving skin barrier and use of such active ingredients to ameliorate, treat or prevent degradation of skin barrier which can lead to decreased aesthetic appearance. Specifically, it is therefore an object of the invention to provide methods for identifying candidates for treatment with an active agent for increasing the aesthetic appearance of skin byimproving skin barrier integrity by increasing synthesis of certain proteins essential for skin barrier function; and / or use of identified actives to increase production of involucrin and / or filaggrin. It is a further object of the invention to improve overall appearance of skin and hair by use of such compositions.

[0014] The foregoing discussion is presented solely to provide a better understanding of the nature of the problems confronting the art and should not be construed in any way as an admission as to prior art nor should the citation of any reference herein be construed as an admission that such reference constitutes “prior art” to the instant application.SUMMARY OF THE INVENTION

[0015] The invention is generally directed to identification and use of an active ingredient, specifically a kombucha extract, an alpha-glucan oligosaccharide, a Thermus thermophilus ferment or a mixtures thereof that is capable of increasing skin barrier protection as assessed by ability to increase certain biomarkers of cell differentiation (filaggrin and involucrin).BRIEF DESCRIPTION OF THE FIGURES

[0016] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0017] In the drawings, which are not necessarily drawn to scale, like numerals describe substantially similar components throughout the several views. Like numerals having different letter suffixes represent different instances of substantially similar components. The drawings illustrate generally, by way of example, but not by way of limitation, various aspects of the present invention.

[0018] FIG. 1 shows the results of studies conducted on skin explants using kombucha extract 0.5% and assaying respectively for filaggrin and involucrin synthesis.

[0019] FIG. 2 shows the results of studies conducted on skin explants using Alpha- Glucan Oligosaccharide extract 0.5% and assaying for filaggrin synthesis.

[0020] FIG. 3 shows the results of studies conducted on skin explants using Alpha- Glucan Oligosaccharide extract 0.5% and assaying for involucrin synthesis.DETAILED DESCRIPTION OF THE INVENTION

[0021] Reference will now be made in detail to certain aspects of the disclosed subject matter, examples of which are illustrated in part in the accompanying drawings. While the disclosed subject matter will be described in conjunction with the enumerated claims, it will be understood that the exemplified subject matter is not intended to limit the claims to the disclosed subject matter.

[0022] Throughout this document, values expressed in a range format should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. For example, a range of “about 0.1% to about 5%” or “about 0.1% to 5%” should be interpreted to include not just about 0.1% to about 5%, but also the individual values (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the indicated range. The statement “about X to Y” has the same meaning as “about X to about Y,” unless indicated otherwise. Likewise, the statement “about X, Y, or about Z” has the same meaning as “about X, about Y, or about Z,” unless indicated otherwise.

[0023] In this document, the terms “a,” “an,” or “the” are used to include one or more than one unless the context clearly dictates otherwise. The term “or” is used to refer to a nonexclusive “or” unless otherwise indicated. The statement “at least one of A and B” or “at least one of A or B” has the same meaning as “A, B, or A and B .” In addition, it is to be understood that the phraseology or terminology employed herein, and not otherwise defined, is for the purpose of description only and not of limitation. Any use of section headings is intended to aid reading of the document and is not to be interpreted as limiting; information that is relevant to a section heading may occur within or outside of that particular section. A comma can be used as a delimiter or digit group separator to the left or right of a decimal mark; for example, “0.000,1” is equivalent to “0.0001.” All publications, patents, and patent documents referred to in this document are incorporated by reference herein in their entirety, as though individually incorporated by reference. In the event of inconsistent usages between this document and those documents so incorporated by reference, the usage in the incorporated reference should be considered supplementary to that of this document; for irreconcilable inconsistencies, the usage in this document controls.

[0024] In the methods described herein, the acts can be carried out in any order without departing from the principles of the invention, except when a temporal or operational sequence is explicitly recited. Furthermore, specified acts can be carried out concurrently unless explicitclaim language recites that they be carried out separately. For example, a claimed act of doing X and a claimed act of doing Y can be conducted simultaneously within a single operation, and the resulting process will fall within the literal scope of the claimed process.

[0025] The term “about” as used herein can allow for a degree of variability in a value or range, for example, within 10%, within 5%, or within 1% of a stated value or of a stated limit of a range, and includes the exact stated value or range. The term “substantially” as used herein refers to a majority of, or mostly, as in at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999% or more, or 100%. The term “substantially free of’ as used herein can mean having none or having a trivial amount of, such that the amount of material present does not affect the material properties of the composition including the material, such that about 0 wt% to about 5 wt% of the composition is the material, or about 0 wt% to about 1 wt%, or about 5 wt% or less, or less than or equal to about 4.5 wt%, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.01, or about 0.001 wt% or less, or about 0 wt%.

[0026] The term “improving aesthetic appearance” as used herein means be an improvement of any attribute or characteristic of skin, including without limitation The term “improving aesthetic appearance” as used herein means be an improvement of any attribute or characteristic of skin, including without limitation Generally, the improvement in the condition and / or aesthetic appearance is selected from the group consisting of: reducing dermatological signs of chronological aging, photo-aging, hormonal aging, and / or actinic aging; preventing and / or reducing the appearance of lines and / or wrinkles; reducing the noticeability of facial lines and wrinkles, facial wrinkles on the cheeks, forehead, perpendicular wrinkles between the eyes, horizontal wrinkles above the eyes, and around the mouth, marionette lines, and particularly deep wrinkles or creases; preventing, reducing, and / or diminishing the appearance and / or depth of lines and / or wrinkles; improving the appearance of suborbital lines and / or periorbital lines; reducing the appearance of crow's feet; rejuvenating and / or revitalizing skin, particularly aging skin; reducing skin fragility; preventing and / or reversing of loss of glycosaminoglycans and / or collagen; ameliorating the effects of estrogen imbalance; preventing skin atrophy; preventing, reducing, and / or treating hyperpigmentation; minimizing skin discoloration; improving skin tone, radiance, clarity and / or tautness; preventing, reducing, and / or ameliorating skin sagging; improving skin firmness, plumpness, suppleness and / or softness; improving procollagen and / or collagen production; improving skin texture and / or promoting texturization; improving skin barrier repair and / or function; improving theappearance of skin contours; restoring skin luster and / or brightness; minimizing dermatological signs of fatigue and / or stress; resisting environmental stress; replenishing ingredients in the skin decreased by aging and / or menopause; improving communication among skin cells; increasing cell proliferation and / or multiplication; increasing skin cell metabolism decreased by aging and / or menopause; retarding cellular aging; improving skin moisturization; enhancing skin thickness; increasing skin elasticity and / or resiliency; enhancing exfoliation; improving microcirculation; decreasing and / or preventing cellulite formation; and any combinations thereof.

[0027] The term “substantially free of’ as used herein can mean having none or having a trivial amount of, such that the amount of material present does not affect the material properties of the composition including the material, such that about 0 wt% to about 5 wt% of the composition is the material, or about 0 wt% to about 1 wt%, or about 5 wt% or less, or less than or equal to about 4.5 wt%, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.01, or about 0.001 wt% or less, or about 0 wt%.

[0028] When referring to a whole (e.g. a composition, a formulation, a process, a system) within the present invention, the term “comprises” and variations thereof as used herein can mean “comprises”, “includes” and “consists of’. The expression “comprises essentially” and variations thereof means “consists to a large extent of’ what is specified and does not exclude the presence in the whole of other components that do not substantially alter its properties in the sense of the present invention.

[0029] An embodiment of the invention utilizes an assay for determining the efficacy of an active ingredient for improving aesthetic appearance relate to improved skin barrier function supported by increased filaggrin and / or involucrin synthesis.

[0030] In one embodiment, an active ingredient identified for improving skin barrier function / skin barrier- associated protein differentiation is an alpha-glucan oligosaccharide and / or kombucha extract. In one embodiment, the kombucha extract is derived from sweet black tea, then bio-augmented through fermentation for one week at 25 - 28 degrees Celsius, during which time the new nutrients are generated- all the vitamins and the really “good stuff’ found in the black tea - are concentrated.

[0031] In one embodiment, the active is used at a concentration of 0,0001 to 20% of the full formula.

[0032] In one embodiment, a method of identifying candidates for treatment with an ingredient capable of improving aesthetic appearance of skin by increasing skin barrier formation as exemplified by increased involucrin and / or filaggrin synthesis is employed.

[0033] The invention is generally directed to identification and use of an active ingredient, specifically a kombucha extract, an alpha-glucan oligosaccharide, a Thermus thermophilus ferment or a mixtures thereof that is capable of increasing skin barrier protection as assessed by ability to increase certain biomarkers of cell differentiation (filaggrin and involucrin).

[0034] Specific mixtures of active ingredients within the scope of the present invention are: i) A Kombucha extract; ii) an alpha-glucan oligosaccharide; iii) a Thermus thermophilus ferment; iv) A Kombucha extract and an alpha-glucan oligosaccharide; v) a kombucha extract, and a Thermus thermophilus ferment; vi) an alpha-glucan oligosaccharide and a Thermus thermophilus ferment; vii) a kombucha extract, an alpha-glucan oligosaccharide and a Thermus thermophilus ferment.

[0035] According to the present invention, the composition can be for topical application, in particular in the form of an oil-in-water emulsion, a water-in-oil emulsion, a multiple emulsion (Water / Oil / Water or Oil / Water / Oil), a microemulsion, a nanoemulsion, a solution, a suspension, a hydrodispersion, a gel, an ointment, a paste, an aerosol foam, a spray, an aqueous gel, a powder, a foundation, a transdermal patch, a cream or a mask. How to prepare a topical cosmetic or dermatological formulation is within the skills of the person skilled in the art, see for example “Cosmetic Formulation: Principles and Practice” by Heather A.E. Benson et al. CRC Press, 2019.

[0036] The formulations can include additional ingredients, such as butters, emollients, emulsifiers, exfoliants, hydrosols, oils, preservatives, solubilizers, surfactants, waxes and UV filters. The specific amount of each of these ingredients to be used in the formulation of the active ingredient(s) according to the present invention can be determined by the skilled person with reference to his common general knowledge.

[0037] In the topical formulations the active agent(s) can be present in an amount of between about 0.001% and about 10%, preferably between about 0.01% and about 1%, more preferably between about 0.1% and about 0.5% based on the total weight of the formulation.Results

[0038] FIG. 1 shows the results of studies conducted on cells utilizing a Kombucha extract yeast extract and assaying for skin barrier maintenance or improvement exemplified by increased involucrin and / or filaggrin synthesis by measuring the amounts of filaggrin and involucrin produced by cells treated and untreated with a kombucha extract. As can be seen in FIG. 1, cells treated with a Kombucha extract exhibited an increased level of involucrin and filaggrin synthesis. Normal human skin explants were treated with the composition for 9 days and incubated at 37°C, 5% CO2. Then filaggrin and involucrin immunostaining were performed on formol-fixed-paraffin embedded skin sections. Filaggrin immunostaining were performed with monoclonal anti-filaggrin antibody and revealed by AlexaFluor488, the nuclei were counterstained using propidium iodide. Involucrin immunostaining were performed with polyclonal anti -involucrin antibody and using a amplifier system avidin / biotin, and revealed by VIP, a substrate of peroxidase. Results were expressed by the surface of the immunostaining.

[0039] FIG. 2 shows the results of studies conducted on skin explants using a alpha- glucamoligosaccahride extract and assaying for skin barrier maintenance or improvement exemplified by increased involucrin and filaggrin synthesis by measuring the amounts of filaggrin and involucrin produced by cells. As can be seen in FIG. 2, cells treated with alphaglucan oligosaccharide exhibited an increased level of filaggrin synthesis. Normal human skin explants were treated with the composition for 9 days and incubated at 37°C, 5% CO2.

[0040] FIG. 3 shows the results of studies conducted on cells using a alpha-glucan oligosaccharide extract and assaying for skin barrier maintenance or improvement exemplified by increased filaggrin synthesis by measuring the amounts of filaggrin produced by cells. As can be seen in FIG. 3, cells treated with alpha-glucan oligosaccharide exhibited an increased level of filaggrin synthesis. Normal human keratinocytes were cultured in KSFM (Keratinocytes serum-free medium). The composition was diluted in the culture medium at 1% or 2.5% and applied on the cells for 72 hours, cells were incubated at 37°C, 5% CO2. Nontreated cells were incubated under the same conditions. At the end of incubation time, the medium was removed, and the cells were rinsed, fixed and permeabilized. Cells were then labeled using a specific primary anti-filaggrin antibody. The primary antibody was revealedusing fluorescent second antibody (Alexa 488) and cell nuclei were stained with Hoechst solution. The fluorescence intensity was then measured to determine the results. The terms and expressions that have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the aspects of the present invention. Thus, it should be understood that although the present invention has been specifically disclosed by specific aspects and optional features, modification and variation of the concepts herein disclosed may be resorted to by those of ordinary skill in the art, and that such modifications and variations are considered to be within the scope of aspects of the present invention.

Claims

CLAIMSWhat is claimed is:

1. A method of improving an aesthetic appearance of skin of a human affected by weakened skin barrier comprising topically applying thereto a therapeutically effective amount of an active agent, wherein the active agent increases synthesis of involucrin and / or filaggrin by greater than 40% when applied ex vivo to human skin keratinocytes at a concentration of at least 0.45% w / w in a cosmetically acceptable vehicle.

2. The method of claim 1, wherein the active agent comprises an extract of Kombucha in combination with and at least one of alpha-glucan oligosaccharide and Thermus thermophilus ferment.

3. The method of claim 2, wherein the active agent comprises: a. Kombucha extract and alpha-glucan oligosaccharide; b. Kombucha extract and Thermus thermophilus ferment; or c. Kombucha extract, alpha-glucan oligosaccharide and Thermus thermophilus ferment.

4. The method of claim 1 , wherein improving the aesthetic appearance of the skin includes reducing and / or preventing the appearance of wrinkles and / or fine lines, improving skin texture, skin tone, pigmentation, skin elasticity and / or signs of ageing and / or reducing hyperhidrosis.

5. A cosmetic or dermatological composition comprising: a. Kombucha extract and alpha-glucan oligosaccharide; b. Kombucha extract and Thermus thermophilus ferment; or c. Kombucha extract, alpha-glucan oligosaccharide and Thermus thermophilus ferment.

6. The cosmetic or dermatological composition according to claim 5 for topical administration.

7. The cosmetic or dermatological composition according to claim 6 in the form of an oil-in-water emulsion, a water-in-oil emulsion, a multiple emulsion (Water / Oil / Water or Oil / Water / Oil), a microemulsion, a nanoemulsion, a solution, a suspension, a hydrodispersion, a gel, an ointment, a paste, an aerosol foam, a spray, an aqueous gel, a powder, a foundation, a transdermal patch, a cream or a mask.

8. The cosmetic or dermatological composition according to claim 7, wherein the Kombucha extract is present in an amount of between about 0.001% and about 10%, preferably between about 0.01% and about 1%, more preferably between about 0.1% and about 0.5% based on the total weight of the formulation.

9. The cosmetic or dermatological composition according to claim 8, wherein the Kombucha extract is present in an amount of about 0.20% to about 3%, preferably about 0.5% based on the total weight of the formulation.

10. The cosmetic or dermatological composition according to claim 9, wherein the alphaglucan oligosaccharide is present in an amount of about 0.25% to about 3%, preferably about 0.5% based on the total weight of the formulation.

11. The cosmetic or dermatological composition according to claim 10, wherein Thermus thermophilus ferment, if present, is present in an amount of about 1% to about 3% based on the total weight of the formulation.

12. A method for screening for putative active agents capable of efficacious improvement of aesthetic appearance of human skin, comprising measuring a relative amount of involucrin and / or filaggrin synthesis in treated and untreated cells; and selecting the putative active agent for use if the putative active agent increases involucrin and / or filaggrin synthesis by at least 40% over baseline.

13. A method for providing a personalized method for improvement of aesthetic appearance of skin, comprising assessing individual skin for relative rate of transepidermal water loss; and suggesting an aesthetic appearance improvement regimen to the individual comprising use of a cosmetic product including an active agent that increases involucrin and / or filaggrin synthesis by at least 40% over baseline if the assessed skin has a rate of transepidermal water loss above 8 g / m2 / hour.

Images