TECHNOLOGICAL FIELD

The invention generally pertains to the field of dermatological and cosmetic compositions using combinations of probiotic and herbal extracts, and specifically to compositions comprising live probiotics and prebiotics and Polygonum plant extracts.

BACKGROUND

The skin microbiome has been attracting increasing attention in recent years, from dermatologists and cosmetologists as one. It is now well established that a balanced skin microbiome in terms of preservation and maintenance of various commensal species composing the skin microbiota, i.e., bacteria, archaea, fungi, yeast, and viruses, is one of the key factors in protecting the skin against environmental and internal insults, and that an imbalance in the normal “healthy” diversity of skin microbiota (dysbiosis) is closely associated with various dermatological disorders and premature skin aging.

The skin is the largest organ with the outermost interface with the environment, whereby it creates multiple ecological niches for various commensal microorganisms. Microbiome, often referred to as “the hidden organ,” is a term denoting a functioning community of such commensal microorganisms at a specific body area of the host, in relation to the entire range of their phylogenetic, genetic, and biochemical components, their networking, and their interaction with the host. Microbiome, and especially the skin microbiome, is highly variable and dynamic structure that changes drastically in the same individual host and between individuals. In the context of skin, differences in skin temperature, humidity, sebum, acidity, and exposure across various skin areas and under various external conditions all contribute to inter- and intra-individual differences in the diversity and composition of individual skin microbiome. Some experts maintain that the diversity of skin microbiome is higher than the one in the gut.

Numerous studies suggested that specific changes in the composition of skin microbiome could be related to various skin conditions, including atopic dermatitis, acne vulgaris, rosacea, seborrheic dermatitis, pityriasis versicolor, psoriasis, lupus erythematosus, diabetic foot ulcer, actinic keratosis, and many others. More generally, it was shown that skin microbiome contributes to the normal functionality of epidermal skin barrier and preservation of skin homeostasis and thereby to healthy and rejuvenated skin appearance. On the other hand, dysbiosis or other deficiencies of skin microbiome were related to aberrant wound healing and increased susceptibility to skin pathogens and other external insults. Immunological studies showed that skin microbiota plays a critical role in shaping the host local immunity via ongoing dialog with resident dendritic cells and activation of innate and adaptive immunity, involving the production of various cytokines and antimicrobial peptides, activation of complement cascade, and signaling and homing of T cells, etc. In addition, the commensal skin microbiota, bacteria and fungi, can prevent the establishment of potential pathogens by quorum-sensing, competition on resources and production of antibiotics. One example is lugdunin - an effective antibiotic produced by Staphylococcus lugdunensis that makes part of the human skin microbiome.

There is a multiplicity of factors contributing to inter- and intra-individual variability in the human skin microbiome, including ethnicity, aging, gender and lifestyle, and physiological differences such as hormone metabolism, perspiration rate and skin pH. In addition, environmental conditions such as exposure to pollution and UV radiation can cause instability and dysregulation of the human skin microbiome, as well as common skin conditions such as acne and seborrhea, and less common skin pathologies such as vitiligo or lupus. It was well demonstrated that certain types of cosmetics and topical and oral medications, and especially antibiotics, can influence commensal microbiota.

With this understanding, pharma and cosmetic industries have started to change their approaches towards design and development of products with pre-, pro- and post- biotic components so as to provide support and protection to the human skin microbiome. Probiotics are mixes of microorganisms with benefits for healthy microbiome (e.g., bacteria or yeast). Prebio tics are compounds that stimulate microbial growth (e.g., carbohydrates) Synbiotics are mixes of prebiotics and probiotics. Postbio tics are additional components derived from live bacteria that contribute to probiotic activity (e.g., amino acids, antimicrobial peptides). In most countries probiotics are regulated under the umbrella of foods and supplements. The main challenge remains in the ability to provide live and sustainable preparations of probiotics that could be incorporated into pharmaceutical and cosmetic formulations without compromising their viability and shelf-life and to ensure their successful delivery and activity in situ upon topical application onto the skin. The existing cosmetic products, for example, often use bacterial cell lysates and not live bacteria, and thus are essentially deprived of all the desired benefits of genuine probiotics. Certain cosmetic and dermatological compositions with probiotics were reported in patent literature, most of them using representatives of Lactobacillus and/or Bifidobacteria species (e.g., W02017063066A1, US10695386B2, US10238597B2, US20080107699A1, EP3727594A1, JP2009503042A, EP2704704B1, US11007137B2). While these two genera may have well documented benefits for the gut microbiome, their role in the skin microbiome is much less established as well as their use and delivery with topical applications. Another approach was suggested in EP3210612B 1 and EP0975227A1, describing cosmetic compositions with certain Bacillus strains in the form of spores and a prebiotic sugar, but without significant data on their safety and efficacy.

Overall, the existing approaches are incomplete and leave much room for improvement. Therefore, there is an unmet need to find effective and safe probiotic constructs that would match the specific conditions of the human skin microbiome and would further account for its vast inter- and intra- individual variability. In other words, there is a necessity to find an improved fit-all skin probiotics that would be sufficiently effective and safe so as to be applicable for rarer dermatological problems and also for more common cosmetic skin conditions.

GENERAL DESCRIPTION

Polygonum is a flowering plant in the buckwheat-knotweed family Polygonaceae. It is being extensively used in traditional medicines for a variety of indications, including dizziness, soreness of back and knees, premature ejaculation, fatigues, atherosclerosis, high cholesterol, insomnia, menstrual problems, malaria, and many other conditions. It is widespread in temperate regions across many countries, with about 130 known species. A strain of Polygonum that is prevalent in Eurasia, Polygonum aviculare, proved to be particularly beneficial for skin conditions and atopic deficiencies. It was shown to possess a range of beneficial activities, starting from antiviral, antibacterial and anti-aging effects, and prevention of various skin infections, and up to restoration of skin moisturization and texture and preservation of a good skin complexion in general. More recent studies showed that it exerts curative effects on various types of eczema and itching and prevents or attenuates the exacerbation and recurrence of atopic dermatitis and other inflammatory skin conditions. Extracts of P. aviculare were shown to contain a particularly high content of active alkaloids and polyphenols, including flavonoids, terpenoids, phytoestrogens, quinones. Examples of such phytochemicals are shown in Table 1. Table 1. Active phytochemicals detected in the extracts of P. aviculare

An earlier study by the inventors has demonstrated several specific effects of the P. aviculare extract that make it particularly advantageous for dermatological and cosmetic applications, including prevention or effective inhibition of mast-cell mediated cutaneous anaphylaxis in vivo, effective scavenging of free radicals, and significant bactericidal and fungicidal effects across wide range of skin pathogens (EXAMPLE 1).

The notion of importance of probiotics and synbiotics in supporting the integrity of skin barrier functionality and maintaining normal skin homeostasis and skin microbiome was discussed above. A specific synbiotic preparation composed of a mix of spores of four specific Bacilli strains, i.e., B. subtilis, B. licheniformis, B. megaterium and B. amyloliquefaciens, and the prebiotic sugar inulin was reported in the EP3210612B 1 patent (Synbio®). A preliminary study has shown that this specific preparation can effectively reduce proinflammatory responses by the decreased level of expression of several key proinflammatory cytokines, IL-ip, IL-8 and TNF-a (data not shown).

The invention stems from more recent surprising findings by the inventors in a reconstructed skin in vitro model that was subjected to global transcriptome analysis with the assistance of bioinformatic tools, showing that a combination of Polygonum extract and Synbio® possesses new biological properties that are essentially distinct from the properties of the two components. A pilot study using this advanced molecular approach suggested that the effects of single components and the combination of both components differed strikingly by qualitative and quantitative parameters of the induced gene expression, which was not restricted to individual genes but rather reflected much broader changes in global gene expression (EXAMPLE 2). Using advanced computational methods, the inventors could show that the induced changes in gene expression could be mapped to specific functional signaling pathways, and that the single and the combination preparations were further distinct by the up or down-regulation of specific molecular pathways. Importantly, the results suggested the involvement of pathways responsible for the regulation of cell cycle and pluripotency and differentiation of keratinocytes, and pathways regulating biosynthesis and metabolism of some important epidermal and dermal components, such as retinol, elastin and collagen, and mediators of radical scavenging (EXAMPLE 2).

The studies on the level of skin transcriptome should be further corroborated on the level of proteomics. Nonetheless, they are sufficiently informative as to the distinctive biological properties of the combined preparation of Polygonum extract and Synbio® as opposed to the properties of its single components. They are further informative as to potential benefits of the combined preparations for alleviation and treatment of various skin conditions, and more generally, for restoration and maintenance of skin homeostasis and skin microbiome.

Ultimately, the effectiveness of a probiotic preparation is highly dependent on the ability to preserve the probiotic bacteria in a specific formulation and the ability to evoke bacterial germination upon topical application onto the skin. Synbiotic preparations, and particularly those including bacterial spores, should in theory comply with both these requirements, assuming prolonged stability of spores and activation of bacterial germination by prebiotic sugar. The question of effectiveness of the presently used synbiotics was addressed in a pilot study of healthy volunteers, daily applying a cream formulation with Synbio® over a 10-day period and testing bacterial viability in topicals samples of these individuals. Surprisingly, live Bacilli bacteria were detected from the initial application and during the entire study period and up to 10 days after the final application, suggesting that the probiotic bacteria remained viable in situ for a substantial period. Further analysis of the samples by 16S rRNA sequencing, and phylogenetic analysis of bacterial diversity, indicated that apart from the introduced Bacilli strains all other bacterial species were characteristic of a normal healthy skin microbiome, and that in most individuals the detected changes were mild and not disruptive (EXAMPLE 3).

Altogether, these results provided proof of concept for potential advantages of preparations combining Polygonum extracts and Synbio® in the context of skin and their potential utility as leave-on products for cosmetic and dermatological uses. Several prototype cosmetic formulations have been produced so far based on P. aviculare extract and the Bacilli-based synbiotics containing a mix of B. subtilis, B. licheniformis, B. megaterium and B. amyloliquefaciens spores and inulin (Synbio®) (EXAMPLE 4). These formulations were further subjected to various dermatological tests, including antimicrobial testing, skin sensitivity testing and stability studies, following the accepted regulatory guidelines. Preliminary data from these experiments indicate that all the produced formulations were compatible with sensitive skin by the skin sensitization testing, and further that all of them in all storage conditions (at 5°C, 20°C, 30°C and 45 °C) preserved the initial count of viable probiotic bacteria after maximum storage period (3 months). According to recognized standards of stability testing, storage at 45 °C for 8 weeks (2 months) is equivalent to storage at RT for one year (EXAMPLE 5).

In summary, the invention provides a new line of products based on combinations of Polygonum extracts and Bacilli-based synbiotics that proved to be safe and efficient for cosmetic and dermatological uses. The combinations of the invention can be formulated into gels, lotions, creams, sprayable liquids, ointments, powders, or semi-sold formulations. They can be further incorporated into various matrixes and films and provided in the form of dermal patches.

Being classified under the umbrella of food supplements, i.e., generally safe (GRAS), it is projected that the compositions and formulations of the invention can serve both for prevention and treatment. Relying on the present molecular data, they can be effective for alleviation of a wide range of temporary and chronic skin conditions, which in the most general terms can be referred to as clinical and preclinical conditions resulting from disruption of skin homeostasis, and more specifically conditions such as skin infections, skin inflammations, allergies, rashes, erythema, in promoting wound healing, protecting against photoinduced, oxidative and pollution damages, skin drying, discoloration, wrinkling, and skin aging in general. In terms of cosmetic applications, the compositions and formulations of the invention can be articulated in terms of facilitators or inducers of a healthy complexion and rejuvenated appearance of the skin. Moreover, unlike with the products using Lactobacillus-based probiotics, the products derived from the compositions and formulations of the invention meet the conditions of vegan cosmetics. Today, they are undergoing a process of registration under the trademark “PolyBiome”. BRIEF DESCRIPTION OF THE DRAWINGS

To better understand the subject matter and to exemplify how it may be carried out in practice, embodiments will now be described by way of non-limiting examples with reference to the following drawings.

Fig. 1A illustrates the antiallergic effect of P. aviculare extract as revealed in the passive cutaneous anaphylaxis (PCA) model in mice, using anti-DNP-BSA specific IgE (0.5 pg) and DNP-BSA antigen with Evans blue (an indicator of mast cell reaction). The extract was administered topically (100-1000 mg/kg) Ih before antigen exposure, diphenhydramine (DPH) was used as positive control. Figure shows that Polygonum extract inhibited PCA in a dose dependent manner.

Fig. IB illustrates the antioxidant effect of P. aviculare extract by NBT assay (nitroblue tetrazolium assay) in vitro. Cells were treated with the extract (2.5-100 pm/ml) (A) compared to cells treated with catechin as positive control (□), H2O2 measurements were obtained from five independent experiments. Figure shows that Polygonum extract effectively inhibited free radical activity at the concentration as low as 2.5 pm/ml (60% inhibition), reaching a complete inhibition (100% inhibition) at concentrations of 5 pm/ml and higher.

Fig. 2A illustrates the dissimilarities of gene expression induced by single preparations of synbiotics (Syn, Synbio®) or P. aviculare extract (Poly) and combinations of Syn and Poly in various proportions (50_Syn_50_Poly and 33_Syn_67_Poly) as revealed in a reconstructed skin in vitro model by microarray analysis and computational comparisons. Figure shows MDS plot (multidimensional scaling) of calculated dissimilarity (distance) for pairwise comparisons of individual signals on the arrays corresponding to treatments with single and combination preparations and combination preparations with different proportions of Syn and Poly, suggesting marked differences of gene expression in various treatments.

Fig. 2B shows MDS plot that further includes comparisons between treatments with combination preparations normalized to single preparations. Figure shows that the changes in gene expression in combination preparations are distinct from changes in single preparations, and that the relative representation of single components (ratios 1:1 or 1:2) further contributes to these dissimilarities. Fig. 3 illustrates the effect of the 50_Syn_50_Poly preparation (ratio 1:1) on changes in gene expression by the number of up- and down-regulated and unchanged genes, calculating the log fold change (log FC) of each gene in treated vs control groups and adjusted log FC relative to the median expression from the array. A two-fold change in the adjusted log FC was used as a cutoff for selection of up- and down-regulated genes.

Fig. 4 is another presentation of the same analysis by MA plot to illustrate the magnitude of changes (fold change vs no change) and the number of involved genes.

DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS

In most general terms, the invention provides a natural-based solution to problems of the skin stemming from impairments of skin homeostasis or disruption of skin barrier by reconstituting and reinforcing healthy skin homeostasis and healthy skin microbiome.

In one of its main aspects the invention provides compositions and formulations, these terms herein are interchangeable, comprising extracts of plants of the genus Polygonum and certain preparations of probiotics and prebiotics, the combination of these two latter is referred to herein as synbiotics. Synbiotics are distinct by the type of the probiotic component and the prebiotic component. The general advantage of synbiotics is in facilitating the germination or viability of the probiotic component upon application of the formulation onto the skin.

In some embodiments the prebiotic component can be a prebiotic sugar, which is an effective inducer of germination of bacteria and yeast.

In some embodiments the probiotic component can be a mix of live bacteria or yeast. In such cases the prebiotic component or the prebiotic sugar can be an optional component since live probiotics, as opposed to spores, can easily move into a vegetative phase upon topical application of the formulation onto the skin.

In some embodiments the probiotic component can be a mix of bacterial or yeast spores, i.e., dormant probiotics. In such cases the probiotic sugar is required for triggering the spores to move into a vegetative phase and for inducing their germination in situ. This type of synbiotics is advantageous in terms of preservation of the probiotic component.

In some embodiments compositions of the invention can be topical compositions, other words, can be formulated so as to permit direct application thereof onto the skin. The methods of making topical formulations in terms of choice of excipients, stabilizers, skin absorption enhancers, etc., would be well known to a skilled practitioner in the field. Regarding specific components, the invention uses extracts of plants belonging to the genus Polygonum (family Polygonaceae), which includes about 130 species that are distributed worldwide, predominantly in temperate climates. The term “ Polygonum" encompasses herein alternative names such as knotweed, knotgrass, bistort, tearthumb, mile-a-minute, all are perennial flowering plants of herbaceous or woody nature. The term “plant” encompasses herein any part of a plant.

In some embodiments the plant can be of the species Polygonum aviculare, also known as prostrate knotweed, birdweed, pigweed and lowgrass, which encompasses herein all recognized morphological variations and different sub-species of these plants.

The plant extract can be prepared by various known procedures. Non-limiting examples of extraction methods that can be applicable to the invention are steam distillation, filtration, maceration, digestion, decoction, infusion, percolation, Soxhlet extraction using solvents such as acetone, hexane and dichloromethane (DCM), supercritical fluid extraction, supercritical CO2 extraction, ultrasound-assisted, microwave-assisted extractions, and combinations of the above methods, and also extraction by organic solvents such as ethanol, hexane, diethyl ether, chloroform and mixtures thereof in various proportions.

In some embodiments the Polygonum extracts can be prepared by immersion of the plant or a part thereof in a mixture of water, oil and Vaseline (an optional component), which is then boiled, soaked, infused and filtered to obtain a clear extract enriched with the original phytochemicals.

In some embodiments the Polygonum extracts of the invention can be obtained by boiling the Polygonum plant in a mixture comprising water and oil, soaking the boiled mixture, and filtering the mixture to obtain a clear extract.

In some embodiments the Polygonum extracts of the invention can be obtained by steam exposure of the Polygonum plant in a mixture comprising water and oil, soaking the mixture, and filtering the mixture to obtain a clear extract.

In some embodiments the Polygonum extracts of the invention can comprise substantial amounts of water, or more specifically up they can comprise up to 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% and up to 99.99% water as measured by units of weight or volume (w/v, v/v or w/w). In some embodiments the Polygonum extracts can comprise water in the range of 1-5%, 5-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%, 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, 75-80%, 80-85%, 85-90%, 90-95% and 95- 99.99% as measured by units of weight or volume (w/v, v/v or w/w).

In some embodiments the compositions of the invention can comprise more than one type of Polygonum extract or a mix of extracts obtained from more than one species of Polygonum plant.

Regarding the probiotic component, in some embodiments the probiotic component can be yeast. Yeast can exist in vegetative and dormant spore forms. Some examples of yeasts that are used in cosmetic products are Saccharomyces cerevisiae and Torula yeast.

In some embodiments the probiotic component can be bacteria. Bacillus strains, for example, can exist in both vegetative and spore forms. They have been recognized as useful probiotics, predominantly in the context of gut, due to exceptionally high stability under various conditions, and additional benefits.

In some embodiments the compositions of the invention can comprise bacterial spores of one or more Bacillus spp., e.g., 1, 2, 3 or 4 Bacillus spp or more.

In some embodiments the Bacillus spp. can be one of the following Bacillus spp: B. subtilis, B. licheniformis, B. megaterium, B. amyloliquefaciens .

In some embodiments the compositions can comprise one out of four Bacillus spp. as above, i.e., B. subtilis, B. licheniformis, B. megaterium or B. amyloliquefaciens.

In some embodiments they can comprise two out of four Bacillus spp. as above, in various combinations.

In some embodiments they can comprise three out of four Bacillus spp. as above, in various combinations.

In some embodiments they can comprise all four Bacillus spp. as above, i.e., B. subtilis, B. licheniformis, B. megaterium and B. amyloliquefaciens.

The relevant prebiotic sugars are generally oligosaccharides, which are a type of carbohydrate composed of a relatively small number of monosaccharide units. Nonlimiting examples of such sugars are fructo-oligosaccharides, galactooligosaccharides, and human milk oligosaccharides. The general property of these compounds is that they stimulate the growth of the probiotic component, and specifically bacteria. In some embodiments the compositions of the invention can comprise one or more probiotic sugars from natural sources.

In some embodiments the compositions can comprise the least one probiotic sugar which is inulin. Inulin is a naturally occurring oligosaccharide produced by certain plants. It belongs to the family of fructooligosaccharides, it is indigestible and non-absorbable.

In some embodiments the compositions can comprise postbiotics, which are bioactive compounds that support the growth of probiotic bacteria such fatty acids, amino acids and nutrients.

One of the specific features of the present compositions is rooted in the ratio between the two main active components, i.e., the Polygonum extracts and the synbiotics. Under the broadest definition, the compositions of the invention can comprise the Polygonum extracts and the synbiotics at a ratio in the range between about 10,000:1 and about 1 : 10,000, respectively, or more specifically at a ratio in the ranges of about 10,000: 1 to 1:10,000, about 9,000:1 to about 1:9,000, about 8,000:1 to about 1:8,000, about 7,000:1 to about 1:7,000, about 6,000:1 to about 1:6,000, about 5,000:1 to about 1:5,000, about 4,000:1 to about 1:4,000, about 3,000:1 to about 1:3,000, about 2,000:1 to about 1:2,000, about 1,000:1 to about 1:1,000, about 900:1 to about 1:900, about 800:1 to about 1:800, about 700:1 to about 1:700, about 600:1 to about 1:600, about 500:1 to about 1:500, about 400:1 to about 1:400, about 300:1 to about 1:300, about 200:1 to about 1:200, about 100:1 to about 1:100, about 90:1 to about 1:90, about 80:1 to about 1:80, about 70:1 to about 1:70, about 60:1 to about 1:60, about 50:1 to about 1:50, about 40:1 to about 1:40, about 30:1 to about 1:30, about 20:1 to about 1:20, about 10:1 to about 1:10, about 9:1 to about 1:9, about 8:1 to about 1:8, about 7:1 to about 1:7, about 6:1 to about 1:6, about 5:1 to about 1:5, about 4:1 to about 1:4, about 3:1 to about 1:3, about 2:1 to about 1:2 and about 1:1, respectively, as measured by units of weight or volume.

In some embodiments the compositions can comprise the Polygonum extracts and the synbiotics at a ratio in the range between about 10,000:1 and about 1:1, respectively, or more specifically at a ratio in the ranges of about 10,000:1 to 1:1, about 9,000:1 to about 1:1, about 8,000:1 to about 1:1, about 7,000:1 to about 1:1, about 6,000:1 to about 1:1, about 5,000:1 to about 1:1, about 4,000:1 to about 1:1, about 3,000:1 to about 1:1, about 2,000:1 to about 1:1, about 1,000:1 to about 1:1, about 900:1 to about 1:1, about 800:1 to about 1:1, about 700:1 to about 1:1, about 600:1 to about 1:1, about 500:1 to about 1:1, about 400:1 to about 1:1, about 300:1 to about 1:1, about 200:1 to about 1:1, about 100:1 to about 1:1, about 90:1 to about 1:1, about 80:1 to about 1:1, about 70:1 to about 1:1, about 60:1 to about 1:1, about 50:1 to about 1:1, about 40:1 to about 1:1, about 30: 1 to about 1:1, about 20: 1 to about 1:1, about 10: 1 to about 1:1, about 9: 1 to about 1:1, about 8:1 to about 1:1, about 7:1 to about 1:1, about 6:1 to about 1:1, about 5:1 to about 1:1, about 4:1 to about 1:1, about 3:1 to about 1:1, about 2:1 to about 1:1 and about 1:1, respectively, as measured by unit of weights or volume.

In some embodiments the compositions can comprise the Polygonum extracts and the synbiotics at a ratio in the range between about 1,000:1 and about 1:1, respectively, as measured by units of weight or volume.

In some embodiments the compositions can comprise the Polygonum extracts and the synbiotics at a ratio in the range between about 100:1 and about 1:1, respectively, as measured by units of weight or volume.

In some embodiments the compositions can comprise the Polygonum extracts and the synbiotics at a ratio in the range between about 10:1 and about 1:1, respectively, as measured by units of weight or volume.

In some embodiments the compositions can comprise the Polygonum extracts and the synbiotics at a ratio of about 7:3, 3:2, 3:1, 2:1 or 1:1, respectively, as measured by units of weight or volume.

In some embodiments the compositions can comprise the Polygonum extracts and the synbiotics at a ratio of about 2.5:1, 2:1, 1.5:1 or 1:1, respectively, as measured by units of weight or volume.

In some embodiments the compositions can comprise the Polygonum extracts and the synbiotics at a ratio of about 2:1 or 1:1, respectively, as measured by units of weight or volume.

As the Polygonum extracts can comprise substantial amounts of water, in some embodiments the Polygonum extracts can constitute more than about 10% of the composition, or more specifically more than about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% and 95% of the composition, as measured by units of weight or volume (w/v, v/v or w/w).

In some embodiments the Polygonum extracts can constitute more than about 10% of the composition (w/v), or more specifically more than about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% and 95% of the composition (w/v). In some embodiments the synbiotics can be diluted in a mixture of water and glycerin to the consistency of a liquid blend.

In some embodiments said liquid blend can comprise water at a concentration in the range between of about 0.1-5% of the liquid blend, or water at a concentration in the ranges of about 0.1-0.5%, 0.5-1%, 1-1.5%, 1.5-2%, 2-2.5%, 2.5-3%, 3-3.5%, 3.5-4%, 4- 4.5% and 4.5-5% of the liquid blend, and glycerin at a concentration in the range between about 1 %- 10%, or glycerin at a concentration in the ranges of about 1-2%, 2-3%, 3-4%, 4-5%, 5-6%, 6-7%, 7-8%, 8-9% and 9-10% of the liquid blend, as measured by units of weight or volume (w/v, v/v or w/w).

In some embodiments said liquid blend can comprise water at the concentration of about 0.5% and glycerin at the concentration of about 6%, as measured by units of weight or volume (w/v, v/v or w/w).

In some embodiments the diluted synbiotics can constitute about 0.1% to about 10% of the composition, or more specifically in the ranges of about 0.1-0.5%, 0.5-1%, 1- 1.5%, 1.5-2%, 2-2.5%, 2.5-3%, 3-3.5%, 3.5-4%, 4-4.5%, 4.5-5%, 5-5.5%, 5.5-6%, 6- 6.5%, 6.5-7%, 7-7.5%, 7.5-8%, 8-9.5% and 9.5-10% of the composition, as measured by units of weight or volume (w/v, v/v or w/w).

In some embodiments the diluted synbiotics can constitute about 0.1% to about 5% of the composition, as measured by units of weight or volume (w/v, v/v or w/w).

In some embodiments the compositions can be further formulated as a micro- or a nano-particulate matter or encapsulated into micro- or nanoparticles.

In some embodiments the compositions can further comprise at least one additional therapeutic or cosmetic active, a mineral, a vitamin or a nutrient. Non-limiting examples are vitamins A, D, E and K that are common in cosmetic compositions, and the conventional antiviral, antibacterial and anti-inflammatory drugs that included in many dermatological formulations.

In some embodiments the compositions can further comprise various humectants, surfactants, solvents, emulsifiers, etc., to achieve desired consistency and texture. In other words, the compositions can comprise one or more agents from one or more of following groups: humectants, surfactants, emulsifiers, thickeners, preservatives, coloring agents, perfumes, or any combination thereof.

In some embodiments the compositions can further comprise enzymes, phytochemicals, vitamins, antioxidants, fragrance substances and essential oils. Another important feature of the present compositions is revealed in the prolonged stability of the two active components, the Polygonum extracts and the synbiotics. Stability of the Polygonum component can be evaluated by measuring the content or concentration of alkaloids and/or polyphenols at various time points using HPLC or analogous methods. Stability of the synbiotics component in the course time can be evaluated by various bacterial viability assays (e.g., number of CFU (colony formation units) by plating or flow cytometry or MTT assay, and fluorescence staining methods).

In some embodiments the compositions of the invention can have stability for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months and more at room temperature (RT).

In some embodiments the compositions can have stability for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, years and more at RT.

The compositions can be provided in various forms, including but not limited to gels, lotions, creams, liquids and ointments, or more generally, liquid, semi-liquid, semisolid and solid formulations adapted for topical use.

It is another objective of the invention to provide a range of pharmaceutical, dermatological, and cosmetic products based on the above disclosed compositions and formulations.

One type of product is pharmaceutical or dermatological formulations comprising one or more of the presently disclosed compositions and a pharmaceutically acceptable carrier and/or excipient.

Another type is cosmetic formulations comprising one or more of the presently disclosed compositions and a cosmetically acceptable carrier and/or excipient.

Still another type are dermal patches, matrixes or films comprising or impregnated with one or more of the presently disclosed compositions.

In some embodiments the dermatological and the cosmetic formulations of the invention can be a gel, a lotion, a cream, a serum, a sprayable liquid, an ointment, a powder, a mask, a shampoo or a liquid or semi-liquid soap.

Another important feature of the present compositions and formulations is revealed in their ability to provide prevention, treatment or alleviation of disorders or conditions resulting from disruption of skin homeostasis and/or skin microbiome. As has been demonstrated by the examples in this application, the effects of the compositions of the invention are apparent at the molecular and cellular levels in cellular models closely mimicking the architecture and composition of the skin epidermis. In some embodiments the effect of the compositions and formulations of the invention can be revealed by the detection of expression changes in at least one gene (i.e., biomarker), including over (up-regulated) and under (down-regulated) expression using various RNA-based and protein -based methods such quantitative PCR and proteomics.

In some embodiments the effect of the present compositions and formulations can be revealed by the detection of changes in expression of at least one gene (biomarker) related along specific molecular pathways, including but not limited to pathways related to TNF signaling, NF-KB signaling, WNT signaling, proteasome and cell cycle pathways, and pathways responsible for retinol, elastin and collagen metabolism.

For example, the compositions and formulations of the invention can induce changes in the expression of one or more of the following biomarkers:

In some embodiments the compositions and formulations can induce changes in the expression of biomarkers responsible for immunological response, such as cytokines TNFa, TNFp, and interleukins IL6, IL8, ILla, ILlp and IL17.

In some embodiments the compositions and formulations can induce changes in the expression of one or more biomarkers of response to oxidative stress, such as superoxide dismutase (SOD), glutathione peroxidase (GPX), catalase (CAT), glutathione peroxidase (GPX3), thioredoxin (TRX2), cytochrome c peroxidase (CCP1), pentose phosphate pathway enzymes (GND1 and RPE1) and transcription factors for response to oxidative stress (YAP1 and SKN7).

In some embodiments the compositions and formulations can induce changes in the expression of one or more biomarkers of keratinocytes differentiation, such as keratin and filaggrin (FLG).

In some embodiments the compositions and formulations can induce changes in the expression of one or more biomarkers of aging (senescence), such as collagen, elastin, P-galactosidase (P-GAL), Ki-67 nuclear protein, matrix metalloproteinases (MMPs), and pl 6 ^{I K4a} (pl 6) and cyclin-dependent kinase inhibitor (CKVp21) senescence biomarkers.

In some embodiments the compositions and formulations can induce changes in the expression of one or more biomarkers which are growth factors, such as epidermal growth factor (EGF), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), transforming growth factor family (e.g., TGFP, HGNC, TGFpi-3) and growth factors receptors (e.g., EGFR, KGFR, HGFR, TGFR). In some embodiments the compositions and formulations can induce changes in the expression of one or more biomarkers of apoptosis such as caspase 3/7 and caspase 8, for example.

In some embodiments the compositions and formulations can induce changes in the expression of one or more collagen disrupters such as collagenase and/or elastin disrupters such as elastase.

In some embodiments the effect of the compositions and formulations of the invention can be revealed at the level of tissue functionality, such as processes involving neutralization of free radicals and effects of UV, control inflammation, keratinocyte differentiation, using perspective methods that are known in the art.

In some embodiments the effect of the compositions and formulations can be measured in vivo by measuring various physical, biochemical, cellular, immunological and morphological parameters of the skin, tissue and indices of skin homeostasis or skin barrier functionality. The term “skin homeostasis" and “skin barrier" are closely related in the sense of preservation and maintenance of the intrinsic organization of the internal organic milieu as opposed to the erratic external environmental conditions.

In some embodiments the effect of the compositions and formulations can be measured by measuring changes in the diversity and composition of the skin microbiome.

It is another objection of the invention to provide applications and uses of the afore-mentioned compositions and formulations.

In the broadest sense the compositions and formulations of the invention can be used for preventing, treating or alleviating disorders or clinical and preclinical conditions resulting from a disruption of skin homeostasis and/or skin microbiome. These encompass true dermatological disorders with a bona fide clinical manifestation and further common preclinical conditions such as rashes, minor skin inflammations or local infections, and other skin imperfections resulting from natural processes such as drying, discoloration, wrinkling and aging of the skin, and further changes in the diversity and composition of the skin microbiome.

In some embodiments such conditions can be temporary, chronic, or recurrent skin conditions, in terms of timeframe and length of the manifested of symptoms or changes.

In some embodiments such conditions can involve persistent, temporary, or recurrent inflammation of one or more regions of the skin. In some embodiments such conditions can involve persistent, temporary, or recurrent viral, bacterial or fungal infection or an infection by macro-or micro-parasites, as primary or secondary infections. Non-limiting examples are bacteria causing cellulitis, impetigo, and staphylococcal (staph) skin infections, viruses in shingles, warts, and herpes simplex, fungi in athlete's foot and yeast skin infections and parasites such as body lice, head lice, and scabies.

In some embodiments the compositions and formulations of the invention can be for preventing, treating or alleviating symptoms or recurrence of acne, cold sore, blisters, hives, warts, ringworm, impetigo, actinic keratosis, rosacea, a skin allergy, an eczema, contact dermatitis, atopic dermatitis, psoriasis, cellulitis, keratosis pilaris.

A particularly interesting application of the present compositions and formulations is in promoting wound healing and protecting against damages of pollution, irradiation and oxidative stress, and in restoring a healthy complexion and rejuvenated appearance of the skin.

In some embodiments they can be used for improving or promoting wound healing.

In some embodiments they can be used for protecting against photoinduced damage to the skin, including UV damage.

In some embodiments they can be used for protecting against oxidative stress or pollution damage to the skin.

In some embodiments they can be used for protecting against skin aging, skin photoaging, skin inflammaging, skin wrinkling, skin sagging, skin irritation, skin dryness. The term “ inflammaging” implies inflammatory processes related to skin aging.

In some embodiments they can be used for moisturizing, soothing, firming and maintaining a rejuvenated appearance of the skin.

In some embodiments the compositions and formulations of the invention can be used for applications onto sensitive skin.

The invention can be further articulated in terms of analogous cosmetic and dermatological methods and uses. Of particular relevance in this context is the concept of an effective amount of the compositions and formulations. The term “ effective amount” implies herein an amount, a dose or a concentration of active(s) that induces a measurable effect on the molecular, cellular, or tissue levels, or on the level of whole organism. One of the interesting application of the present compositions, formulations and methods is in the field of probiotic-based cosmetics, especially in view of present findings they probiotic component remains viable in situ for a substantial period without interfering or disrupting the normal healthy skin microbiome. In other words, the invention can provide a line of cosmetic leave-on products comprising formulations and compositions of the invention.

The terms “about”, “approximately” , “substantially” permit herein deviations of up to 20% by measurements by weight and/or volume, and specifically up to 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% and 20% by measurements by weight and/or volume.

EXAMPLES

EXAMPLE 1: Therapeutic effects of Polygonum aviculare

P. aviculare extract was prepared by chloroform extraction method. Previous analysis showed that P. aviculare extracts have a particularly high content of flavonoids, terpenoids, phytoestrogens, quinones and other alkaloids, polyphenols (Table 1 above). Total phenolic content of P. aviculare extract can be in the range of 677.4 ± 62.7 mg/g, which is significantly higher than in extracts of aloe vera for example. This study tested several biological activities of the P. aviculare extract using in vivo and in vitro models.

1.1 Antiallergic effect of Polygonum extract

Antiallergic activity of P. aviculare extract was tested in vivo by inducing local mast-cell mediated allergic reaction in mice. Animals were exposed to anti-DNP-BSA (di-nitrophenol conjugated bovine serum albumin) specific IgE (0.5 pg) and further to DNP-BSA antigen containing Evans blue (an indicator of mast cell reaction). The extract was administered topically (100-1000 mg/kg) before antigen exposure (1 h). Diphenhydramine (DPH), an antihistamine drug, was used as positive control. The results are shown in Fig. 1A.

The results show that Polygonum extract inhibited passive cutaneous anaphylaxis (PCA) in a dose dependent manner, suggesting that it exerts its antiallergic effect by preventing the activation of mast cells. 1.2 Antioxidant effect of Polygonum extract

Antioxidant activity P. aviculare extract was tested in vitro by NBT assay (nitroblue tetrazolium assay), measuring the production of superoxide (H2O2) in cells treated with the extract (2.5-100 pm/ml) and using Catechin, a known plant antioxidant, as positive control. Measurements were obtained from five independent experiments. The results (% inhibition) are shown in Fig. IB.

The results show that Polygonum extract effectively inhibited free radical activity at the concentration of 2.5 pm/ml (60% inhibition) and completely inhibited free radical activity at the concentrations of 5 pm/ml and higher (100% inhibition).

1.3 Antimicrobial effect of Polygonum extract

Antimicrobial activity of P. aviculare extract was tested in vitro by antibiotic susceptibility testing (AST) by measuring the diameter of inhibition zone with various Gram-positive and Gram-negative bacteria. Plated bacteria were treated with increasing concentrations (0.4-400 mg/ml) of the extract. The results (mm inhibition zone) are shown in Table 2.

Table 2. Antimicrobial activity of Polygonum extract

The results show that Polygonum extract exerted effective antimicrobial activity on a wide range of Gram-positive (5. aureus, S. pyogenes, B. suhililis) and Gram-negative (E. coli, P. aeruginosa, S. typhi, S. paratyphi, S. flexneri) bacteria in a dose dependent manner. 1.4 Antifungal effect of Polygonum extract

Antifungal activity of P. aviculare extract was tested in vitro by AST in different strains of pathogenic fungi (Aspergillus flavus and Aspergillus fumigatus), applying the extract (0.4-400 mg/ml) and measuring the diameter of inhibition zone. The results are shown in Table 3.

Table 3. Antifungal activity of polygonum extract

A. flavus is the second leading cause of invasive aspergillosis. A. fumigatus causes a spectrum of diseases, e.g., allergic bronchopulmonary aspergillosis, chronic pulmonary aspergillosis and invasive aspergillosis. The results show that Polygonum extract exerted effective antifungal activity on both types of fungi in a dose dependent manner.

EXAMPLE 2: Gene expression analysis of Polygonum extract and Synbiotics

Gene expression studies used a previously described preparation of synbiotics (Synbio® described in EP3210612B 1), containing a mix of spores of four specific Bacillus spp. (B. subtilis, B. licheniformis, B. megaterium and B. amyloliquefaciens) and a prebiotic sugar (inulin), and P. aviculare extract. Synbio (Syn) was dissolved in demineralized water up to bacterial concentration of 10E9 CFU/ml and inulin concentration of 1%. P. aviculare extract (Poly) was prepared by solvent extraction method using a mixture of water, oil, and boiling, soaking, infusion and filtering. The two were combined in ratios of 1 : 1 and 1 :2, respectively, to produce combination preparations (50_Syn_50_Poly and 33_Syn_67_Poly). The effects of the single and combination preparations on gene expression were studied in a 3D reconstituted human skin model in vitro (consisting of epidermis and dermis tissues), using high throughput Affymetrix microarray analysis of total RNA obtained from the skin cells after topically treatment with the preparations and control. 2.1 Quantitative and qualitative analyses of global gene expression

The microarrays were subjected to pairwise comparisons of gene signals in each treatment group vs. control and in combination treatment groups vs. single treatments, while considering significant expression changes as log FC other than 1 (log FC=1 is twofold expression change) and FDR above 0.2 (false discovery rate). The numbers of differentially expressed genes (DEG) detected are shown in Table 4.

Dissimilarities between groups were further evaluated using multidimensional scaling (MDS) plots, calculating dissimilarity (distance) for each pairwise comparison of gene signals on the respective arrays. The results are shown in Figs. 2A-2B, illustrating dissimilarities by the positions of groups on the plot. Figures show clear distinctions between groups treated with the single and combination preparations, and also between groups treated with combination preparations with different proportions of Syn and Poly (SymPoly 1:1 or 1:2).

Subsequent heatmap analysis of the array data regarding 4326 DEG (eight-point comparisons of combination treatments vs. controls and vs. single treatments) revealed significant differences between groups in terms of the number of genes involved, the identity of up-regulated and down regulated genes and the intensity of signals, suggesting qualitative and quantitative differences in global expression. The magnitude of changes is illustrated in Boxplot and MA-plot of in Figs 3-4. Overall, these results were unprecedented and surprising and were further revealing as to the range of implications of topical use combinations of Polygonum extracts and synbiotics. 2.2 Analysis of functional gene sets and gene pathways

GSEA analysis (gene set enrichment analysis) of the microarray data, evaluating the data at the level of gene sets selected by affiliation to known molecular pathways from reference databases (KEGG and WIKI), suggested that the arrays derived from single and combination treatments were further distinct by specific molecular pathways that are relevant to the functionality of the skin.

The most significant changes in gene expression were mapped to pathways related to TNF signaling, NF-KB signaling, WNT signaling, proteasome and cell cycle pathways, and pathways responsible for retinol, elastin and collagen metabolism. The implication of TNF, NF-KB and WNT signaling pathways in the context of skin may have consequences on the capacity of the skin to neutralize free radicals and effects of UV, control inflammation, keratinocyte differentiation, and on the overall capacity of the skin for self-renewal. More generally, these results suggest that the Polygonum extract and synbiotics combination induces significant changes in gene expression along multiple molecular pathways that are likely to impact on skin homeostasis.

EXAMPLE 3: Bacterial survival and microbiome effects of Synbiotics

A commercial cream formulation (an oil in water emulsion), containing 1% Synbio® and the following INCI ingredients: Water, Glycerol, Caprylic/capric triglyceride, Sodium polyacrylate, Ethyl hexyl stearate, Trideceth-6, Phenoxyethanol, Galacto-oligosaccharide, Ethyl hexyl glycerol, and Fragrance.

The cream was applied topically by 10 healthy volunteers, with daily applications over a 10-day period. Bacterial viability was tested in topicals samples obtained from these individuals before and after the initial application, and at various time points during the study and up to 10 days after the final application. The samples were further subjected to 16S rRNA sequencing and phylogenetic analysis of bacterial diversity.

Live bacteria were detected in all topical samples, starting from the initial application and during the entire study period, and up to 10 days after the final application. This is a surprising finding, suggesting that the probiotic bacteria remained viable in situ for a substantial period. In-depth analysis of bacterial genome suggested that, apart from the Bacilli strains specific to Synbio®, all other bacterial species were characteristic of a normal healthy skin microbiome, and that in most individuals the detected changes were mild and not disruptive. EXAMPLE 4: Prototype formulations of Polygonum extracts and Synbiotics

Several prototype formulations have been generated so far basing on P. aviculare extract and the Bacilli-based synbiotics containing a mix of B. subtilis, B. licheniformis, B. megaterium and B. amyloliquefaciens spores and inulin (Synbio®). Examples of such formulations are provided in Table 5.

Table 5. Examples of cosmetic formulation with Polygonum extract and Synbio®

Body Lotion

EXAMPLE 5: Dermatological and stability studies

The cosmetic listed formulations were further subjected to various dermatological tests for regulatory purposes, including microbial challenge testing, probiotic bacterial stability, clinical (human) skin sensitization testing and physico-chemical stability studies.

Skin sensitization testing was performed following the accepted regulatory guidelines for in vivo and in vitro testing. Preliminary data from these experiments indicate that all the formulations were found to be compatible with sensitive skin.

Stability studies of probiotic bacterial in the cosmetic formulations considered CFU (count of colony forming units by plating or flow cytometry) after storage in different conditions, including incubation at 30°C, alternating storage at 5°C and 45°C, and dark storage at 20°C as a reference, for the period of 1 week, 2 weeks, 1 month and 3 months. A CFU drop of < * log unit was considered as stable. Preliminary data from these experiments indicate that all formulations in all storage conditions preserved the initial count of viable probiotic bacteria (CFU=30 per plate) after maximum storage period (3 months). Additional samples are now subjected to longer storage periods of 6 months and 1 year. According to the recognized standards of stability testing, storage at 45 °C for 8 weeks (2 months) is equivalent to storage at RT for one year.