Use of polypodium alpinum and dahurian larch for preventing damage from UVA, UVB, UVC and / or IR rays

Abstract

The present invention relates to a composition comprising or consisting of a dry extract of polypody nigra and a dry extract of Larix melastoma bark, the use thereof for preventing damage from exposure to UVA, UVB, UVC and / or IR rays, and a related oral nutritional medicine or pharmaceutical composition. The invention also relates to a composition comprising a dry extract of polypody nigra and a dry extract of Larix melastoma bark, or a combination consisting of a dry extract of polypody nigra and a dry extract of Larix melastoma bark, the use thereof for preventing damage from exposure to UVA, UVB, UVC and / or IR rays.

Description

Technical Field

[0001] The present invention relates to an association comprising plant extracts capable of helping to protect the skin from the main causes of damage associated with exposure to ultraviolet rays, and related oral nutraceutical or pharmaceutical compositions. Background Art

[0002] The skin is the largest organ in the body and contains approximately 1.5-2.0 m 2 UV exposure is one of the key factors in the onset of several skin conditions such as wrinkles, flaking, dryness, abnormal pigmentation (hypopigmentation or hyperpigmentation), etc.

[0003] The solar UV spectrum can be divided into three parts based on the wavelength of the radiation: short wave (UVC; 100-280nm), medium wave (UVB; 280-320nm) and long wave (UVA; 320-400nm). Each spectrum has characteristic efficiency limits in penetrating the epidermis and dermis of human and mouse skin:

[0004] 1.UVC spectrum (100-280nm) UVC radiation is mostly absorbed by the atmospheric ozone layer and does not normally reach the Earth's surface. These wavelengths have enormous energy and are mutagenic in nature. UVC radiation can penetrate the skin to a depth of about 60-80 microns and can damage DNA molecules.

[0005] 2. UVB spectrum (280-320nm) . UVB radiation accounts for about 5% of the total solar UV radiation and is the main cause of a variety of skin diseases, including non-melanoma and melanoma cancers. UVB radiation can penetrate the skin to a depth of about 160-180 microns. It can pass through the entire epidermis and penetrate into the dermal compartment of human skin. It can induce both direct and indirect adverse biological effects, including induction of oxidative stress, DNA damage, premature skin aging, and various effects on the immune system, which together play an important role in the generation and maintenance of UV-induced neoplasms. Although the skin has a complex defense system composed of enzymes and non-enzymatic components to protect it from these adverse biological effects, excessive UV exposure overwhelms and exhausts the skin's defense system.

[0006] 3. UVA spectrum (320-400nm)UVA radiation comprises the broadest spectrum of solar UV light (90-95%) and is considered the "aging rays". UVA rays penetrate deeper into the epidermis and dermis to a depth of about 1000 microns. Extensive exposure to UVA rays has been shown to lead to the formation of both benign and malignant tumors. Exposure to UVA rays induces the formation of ROS, which can cause damage to cellular macromolecules, cause photoaging and compromise certain immune functions.

[0007] Increased ROS production leads to a depletion of the intracellular antioxidant supply, and all of this causes critical cellular oxidative stress that can cause severe damage to cells exposed to UVB rays. This harmful effect is further enhanced when cells are subjected to high-energy UVB photons that can directly induce oxidative damage to cellular components. In this case, lipid degradation changes the fluidity of the cytoplasmic membrane, leading to cell death. Mitochondria integrate a complex system of apoptotic signaling and survival pathways; changes in their membrane potential are considered an early sign of apoptosis. Similarly, previous results have shown that even UV exposure of less duration than the minimal erythema dose is sufficient to cause DNA damage to skin cells. When not properly repaired, DNA modifications are the first step to mutation, carcinogenesis, and premature aging.

[0008] UVB radiation also activates various redox balance-sensitive transcription factors, including NF-κB. This transcription factor is maintained in the cytoplasm in an inactive form as a heterodimer composed of p50 and p65 subunits. Once NF-κB is activated, the cytoplasmic subunits translocate into the nucleus and interact with DNA sequences at the promoter level of target genes, resulting in upregulation of several proinflammatory proteins and metalloproteinases (MMPs).

[0009] Collagenase and elastase are metalloproteinases and two enzyme families involved in the degradation of proteins of epidermal connective tissue. Collagenase is a transmembrane endopeptidase that destroys the peptide bonds of collagen; elastase is a serine protease that degrades elastin fibers. Since both collagen and elastin determine the mechanical properties of the skin, including elasticity, they are precisely fine-tuned to maintain proper tissue homeostasis: their degradation changes the structure of the dermis. In addition to those already described, the establishment of a cellular environment characterized by inflammation is also one of the cellular mechanisms underlying skin photoaging.

[0010] Following exposure to UV rays, we also see an increase in the production of COX-2, a proinflammatory enzyme involved in the metabolism of arachidonic acid and capable of mediating different inflammatory responses. The peak of expression appears to arrive 24 hours after irradiation and also appears to increase with age, thus supporting chronic low-grade inflammation over time. [Rea G Daré, “Abilities of protocatechuic acid and its alkyl esters, ethyl and heptylprotocatechuates, to counteract UVB-induced oxidative injuries and photoaging in fibroblasts L929 cell line,” Journal of Photochemistry & Photobiology, B: Biology, 2020].

[0011] A large body of clinical and experimental evidence suggests that immune factors contribute to the pathogenesis of UV-induced skin cancer in mice and possibly in humans.

[0012] There is great interest in using chemopreventive agents such as plant polyphenols to inhibit these events in skin exposed to UV rays. Various polyphenols and phytochemicals have shown significant photoprotective effects on the skin (e.g., green tea polyphenols, grape seed proanthocyanidins, resveratrol, silymarin, and genistein, etc.), against skin inflammation, oxidative stress, and DNA damage induced by UV rays. [Jea Nichols, "Skin photoprotection by natural polyphenols: Anti-inflammatory, anti-oxidant and DNA repair mechanisms," Arch Dermatol Res., 2010].

[0013] Polypodium (Polypodium Leucotomos) is a fern native to Central and South America, where it is traditionally used to treat inflammatory skin problems, psoriasis and atopic dermatitis. Its ability to reduce the harmful effects of solar radiation has also been demonstrated in many in vitro studies on animal models as well as in clinical studies, making it of interest not only for the prevention of skin phototoxicity, but also for the prevention and treatment of skin problems with the presence of photosensitivity.

[0014] This type of action is associated with the presence of specific substances, including flavonoids and phenolic acids, which have antioxidant effects and are therefore useful for protecting the skin from damage associated with uncontrolled ROS formation due to exposure to sunlight and in particular to UV radiation [K. Segars, V. McCarver and MRA, "Dermatologic Applications of Polypodium leucotomos: A Literature Review," Journal of Clinical and Aesthetic Dermatology, 2021].

[0015] The action of natural compounds such as polyphenols, carotenoids, vitamins, anthocyanins is of interest in combating UV damage, as they not only act by blocking UV entry into the skin (as topical sunscreens do), but also work in synergy with the latter, having a direct scavenging effect on ROS, and also blocking the signal transduction pathways of ROS. Therefore, it is important to protect the skin even after exposure to UV, as ROS can activate photosensitizing compounds (photosensitizers) and thereby cause greater cell damage. Various antioxidant compounds have been studied for these characteristics, including protocatechuic acid and its alkyl esters. These are compounds present in large quantities in many edible and medicinal plants.

[0016] Polypodium extracts have been tested in vitro on irradiated fibroblasts (L929) and have shown high antioxidant potential. In experimental systems, such compounds have also been shown to inhibit the activity of collagenase and elastase, thereby acting on one of the potential biological mechanisms of skin aging. Protocatechuic acid and its derivatives can absorb UVB radiation, thereby acting as "sunscreens" to reduce the penetration of UV rays in cells and can also at least partially restore the cellular antioxidant defense system (SOD, catalase, GSH). Counteracting oxidative stress means that these compounds can inhibit damage to lipids, mitochondrial membranes and DNA. In addition, PA has been shown to reduce the nuclear translocation of NF-κB, and pre-treatment of cells with PA and its derivatives counteracts the overexpression of COX-2 caused by UV irradiation.

[0017] The protective effects seen so far are also caused by other phenolic compounds, such as chlorogenic acid, ellagic acid, and caffeic acid. Pretreatment of cells with chlorogenic acid before UVB exposure can prevent DNA damage and increase cell viability, and also prevent or limit apoptosis and cell modifications associated with it [JeaWon Cha, "The Polyphenol ChlorogenicAcid Attenuates UVB-mediated Oxidative Stress in Human HaCaT Keratinocytes," Biomol Ther, 2014].

[0018] The effects of chlorogenic acid are also important with respect to UVA damage, highlighting its protective role against photoaging. In irradiated cells, it can stimulate collagen production (and reduce the action of matrix metalloproteinases), maintain cell integrity, and can play a scavenging role against ROS [NEaXue, "Chlorogenic Acid Prevents UVA-Induced Skin Photoaging through Regulating Collagen Metabolism and Apoptosis in Human Dermal Fibroblasts," Int. J. Mol. Sci., 2022].

[0019] One of the most studied extracts for photoprotection is An aqueous extract from the leaves of Polypodium vulgare, which in fact has a large number of in vitro and in vivo studies, has been the subject of patent EP0813405B1.

[0020] In recent studies with cell models, it was also found to be able to induce an increased transcription of the nuclear factor NRF2 and its signaling pathway, which is protective for the cells. Among the targets of NRF2 we found, for example, several enzymes such as catalase, glutathione peroxidase (GPX), NAD(P)H quinone dehydrogenase (NQO1), which support the protection against damage caused by ROS. This protective effect is of interest not only against UV-induced damage, but also against stress associated with exposure to specific pollutants such as fine dust (PM2.5) [PeaDelgado-Wicke, " UpregulatesNRF2Antioxidant Pathway and Protects Keratinocytes from PM2.5-InducedXenotoxic Stress,” Oxidative Medicine and Cellular Longevity, 2020].

[0021] It may also be useful as an adjunct to the treatment of melasma and vitiligo, as it has a protective effect against hyperpigmentation of the skin.

[0022] Larch is a deciduous conifer belonging to the Pinaceae family, widely distributed in the alpine forests of Central Europe, the Alps and the Carpathians, between 180m and 2600m above sea level. Its height varies from 25 to 45m, and its trunk diameter can reach 1m.

[0023] Larix is ​​the only conifer that lights up the high-altitude woods with its golden color in the fall and completely sheds its needles in the winter.

[0024] Due to the durability of the wood of its stem, this plant is mainly used in carpentry, construction and in the marine field [V. Chalupa, "Larch (Larix decidua Mill.)," in Trees III. Biotechnology in Agriculture and Forestry, vol 16., Springer-Verlag Berlin Heidelberg, 1991, pp. 446-470].

[0025] However, there is also some evidence of traditional use of larch bark decoctions as anthelmintics, diuretics, purgatives, for the treatment of respiratory diseases, tuberculosis and rheumatism. For external use, it has been used to treat wounds, insect bites, eczema, psoriasis and as a wound healer. On the other hand, larch resin is an ancient antibacterial drug for indoor and outdoor use called "Venice turpentine" [Lust, The Herb Book: The Most Complete Catalog of Herbs Ever Published, Courier Corporation: Chelmsford, MA, USA., 1974].

[0026] In a study published by Baldan et al. [Baldan V, Sut S, Faggian M, Dalla Gassa E, Ferrari S, De Nadai G, Francescato S, Baratto G, Dall'Acqua S. Larix decidua Barkas a Source of Phytoconstituents: An LC-MS Study. Molecules. 2017 Nov 15; 22(11): 1974. doi: 10.3390 / molecules22111974. PMID: 29140273; PMCID: PMC6150244.], an extraction process starting from European larch (Larix decidua, European larch) using solvents with low environmental impact was reported.

[0027] The obtained extracts were characterized by the content of polyphenols and proanthocyanidins in order to evaluate their antioxidant activity.

[0028] The results of such studies indicate that larch bark is a valuable source of antioxidant compounds such as flavonoids and B-type proanthocyanidins (PACs) and may represent an innovative and sustainable ingredient for pharmaceutical, nutraceutical and cosmetic purposes.

[0029] The applicant filed European Patent Application No. 22173091.4, which claims the use of extracts of European larch for its antibacterial properties in the upper respiratory tract. Summary of the invention

[0030] The applicants have now discovered that a combination of an extract of Polypodium vulgare and an extract of Larix olgensis provides the body with a targeted uptake of substances that can work synergistically with topical sun products for protection against damage induced by sun exposure.

[0031] Such combinations produce a synergistic effect that can be exploited to counteract the damage from exposure to UV rays and the resulting photoinduced skin aging.

[0032] The object of the present invention is therefore the aforementioned combination, comprising or consisting of a dry extract of Polypodium vulgare and a dry extract of Larix olgensis bark, for use in preventing damage from exposure to UVA, UVB, UVC and IR rays. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 : Bioluminescence analysis. The luminescent signal is proportional to the caspase activity (ALU = arbitrary luminescence unit).

[0034] Figure 2 : Immunohistochemical analysis. Quantification of the intensity (total intensity) of involucrin (IVL) expression. DETAILED DESCRIPTION

[0035] For the purpose of this patent application, the expressions "comprising" or "containing" provide the possibility of further components than those explicitly mentioned after such expressions.

[0036] On the contrary, for the purpose of the present invention, the expression "consisting of" excludes the possibility of further components than those explicitly listed after such expression.

[0037] As already stated, the combination objects of the invention are intended for use in preventing damage from exposure to UVA, UVB, UVC and / or IR rays.

[0038] Preferably, the combination objects of the present invention are intended for use in preventing damage from exposure to UVA and UVB rays.

[0039] Preferably, the use of the combination for prophylactic purposes is intended for patients with:

[0040] - Photoaggravated diseases such as lupus erythematosus, lucites, melasma

[0041] - Clear phototype with specific reactivity to sunlight and / or susceptibility to solar urticaria

[0042] - Patients with a family history of skin cancer or who already have a precancerous or cancerous form

[0043] - Chronic inflammatory diseases associated with barrier alterations that benefit from sunlight exposure (atopic dermatitis, psoriasis, acne)

[0044] -Photoaging manifestations.

[0045] Still preferably, the use of the combination for prophylactic purposes is also intended for:

[0046] - Subjects who are undergoing phototherapy to protect against the negative effects of UVB

[0047] - Subjects undergoing phototoxic or photosensitizing drug therapy. In these cases, the combination will not prevent skin reactions, but will increase the minimum phototoxic dose (MPD).

[0048] The combination for use according to the invention is preferably in the form of an oral composition comprising excipients and / or diluents suitable for producing a formulation intended for oral ingestion.

[0049] For the purposes of the present invention, the excipients that can be used are those excipients commonly known to those skilled in the art for preparations of oral forms such as powders, granules, capsules, tablets, solutions or oral suspensions. As non-limiting examples, suitable excipients can be selected from one or more of the excipients listed below:

[0050] a) Diluents such as calcium hydrogen phosphate, microcrystalline cellulose and cellulose derivatives

[0051] b) Thickeners such as hydroxypropyl methylcellulose and cellulose derivatives, gums

[0052] c) Sweeteners such as sucralose, sorbitol, mannitol and other polyols

[0053] d) lubricants such as magnesium stearate, waxes, stearic acid,

[0054] e) Dispersants

[0055] f) Flavoring agents

[0056] g) Adsorbents such as silica, talc, starch

[0057] h) Glidants

[0058] i) Non-stick agents such as talc, colloidal silicon dioxide, corn starch, silicon dioxide

[0059] j) Dyes such as iron oxides, riboflavin, chlorophyll, etc.

[0060] k) Antioxidants

[0061] l) Binders such as starch, gelatin gums, sodium alginate, cellulose derivatives

[0062] m) Disaggregants such as microcrystalline cellulose, starch, cross-linked polyvinylpyrrolidone, alginic acid

[0063] n) Plasticizers such as ethyl cellulose and cellulose derivatives, glycerol and sorbitol

[0064] o) Preservatives such as potassium sorbate and sodium benzoate

[0065] p) Thickener

[0066] q) Emulsifier

[0067] r) Moisturizer.

[0068] Preferably, the oral composition comprising the combination of the invention is in the form of a food supplement, a nutraceutical product or a pharmaceutical product. In each case, the combination of the invention constitutes the active ingredient of the oral composition, or is the only active ingredient of the oral composition.

[0069] For the purposes of the present invention, a "nutraceutical" is defined as a food (or a portion thereof), preferably a portion thereof (particularly the extract subject of the present invention), according to the definition given by Stephan Defelice in 1989, and which has a positive effect on well-being and health, including the prevention and treatment of disease.

[0070] "Drug" product means any substance or combination of substances that appears to have therapeutic or preventive properties for human disease; or further, means any substance or combination of substances that can be used or administered to humans to restore, correct or alter physiological function, to exert a pharmacological, immunological or metabolic effect, or to establish a medical diagnosis.

[0071] For the purposes of the present invention, a food supplement means a preparation falling within the definition of Directive 2002 / 46 / EC and subsequent amendments. In this decree, a food supplement is explicitly defined as: "a food intended to supplement the normal diet and which is a concentrated source of nutrients (such as vitamins and minerals) or other substances having a nutritional or physiological effect (in particular but not limited to amino acids, essential fatty acids, fibers and extracts of plant origin), both as single compounds and as a plurality of compounds, in a predetermined dosage form".

[0072] For the purposes of the present invention, European larch extract (LBE) is a bark extract, preferably obtained from organic bark waste.According to a preferred embodiment, the organic bark waste comprises or consists of industrial waste from the processing of larch wood.

[0073] Still preferably, the bark extract (LBE) of European larch is obtained by extraction with an aqueous alcoholic solvent, preferably ethanol, preferably following the teaching of Baldan et al. [Baldan V, Sut S, Faggian M, Dalla Gassa E, Ferrari S, De Nadai G, Francescato S, Baratto G, Dall'Acqua S. Larix decidua Barkas a Source of Phytoconstituents: An LC-MS Study. Molecules. 2017 Nov 15; 22(11): 1974. doi: 10.3390 / molecules22111974. PMID: 29140273; PMCID: PMC6150244].

[0074] According to a preferred embodiment, the extraction process of the bark of European larch is described in European patent application EP 4 088 730 A1 in the name of the present applicant.

[0075] According to a preferred embodiment, the oral composition comprising the combination of the invention comprises a dry extract of larch bark in an amount comprised between 40 and 90 mg, preferably between 50 and 85 mg, preferably between 60 and 80 mg.

[0076] Preferably, the Polypodium villosum extract is an extract obtained from the rhizomes and / or aerial parts of the plant, preferably characterized by a polyphenol content comprised between 3% and 8%, preferably equal to about 5% by weight (UV detection method).

[0077] Preferably, the extract of Polypodium villosum can be obtained using a process comprising the following steps:

[0078] -Prepare samples of Phlebodium aureum (or Phlebodium aureum)

[0079] - Optionally, pre-treat the sample by washing it with water to remove its coarse impurities

[0080] - Extract the cleaned sample in a hydroalcoholic solvent, preferably 50-80% ethanol, preferably 70%

[0081] - concentrating the extract and optionally drying the extract,

[0082] The process is preferably characterized by a drug / extract ratio comprised between 4:1 and 6:1, preferably 5:1.

[0083] Still preferably, the step of drying the extract of Polypodium villosum comprises the following sub-steps:

[0084] - Adding an inert carrier such as maltodextrin to the concentrated extract

[0085] - The extract with added carrier is spray dried.

[0086] The process preferably comprises the steps of grinding and sieving the dried extract after the extract drying step.

[0087] Still preferably, the dry extract of Polypodium villosum may be present in an amount comprised between 100 mg and 900 mg, preferably between 200 mg and 700 mg, even more preferably between 300 mg and 500 mg.

[0088] The combination for use according to the invention is preferably in the form of an oral composition, preferably administered once a day, and preferably in the evening before bedtime to promote the cell and DNA repair processes that occur at rest.

[0089] The combination of the invention for use purposes may also comprise at least one active ingredient chosen from vitamin C and vitamin B3 or mixtures thereof, whether the composition delivering the combination of the invention is in the form of a nutraceutical or pharmaceutical product or a food supplement.

[0090] Example

[0091] I. Combinations according to the invention

[0092] For illustrative and non-limiting purposes, an example of an oral formulation in the form of a tablet comprising the combination for use according to the invention is given below.

[0093]

[0094] II. In vitro studies: The ability of the combinations of the invention to counteract the primary damage induced by UV rays was evaluated on a model of phototype II reconstructed human epidermis (RHPE).

[0095] II.1 Purpose

[0096] The aim of this study was to evaluate the multifunctional protective effect of the combination of the invention in counteracting the primary damage induced by UV rays in the presence of RHPE type II melanocytes irradiated with 2 UV MED equivalent to a daily exposure on reconstructed human epidermis. Specifically, the effect of the combination was observed in the following aspects:

[0097] -UV-induced inflammatory cascade

[0098] -Integrity of the skin barrier.

[0099] Pigmented tissues were treated overnight in culture medium with (previously determined) non-cytotoxic doses of the following substances:

[0100] (P1) Polypodium cyanus rhizome extract (drug / extract ratio = 5:1; extraction solvent = ethanol 70% / water 30%; maltodextrin carrier 10%; polyphenol titer = 5.0%).

[0101] (P3) A mixture of (P1) an extract of Polypodium vulgare rhizome and (P2) an extract of LARIX DECIDUA L. bark (described in European patent application EP 4 088730 A1 in the name of the present applicant).

[0102] The tissue was then irradiated with UVA+UVB equivalent to 2 MED.

[0103] Samples were collected 4 hours and 24 hours after exposure to UV rays in the presence of fresh medium containing the element in question.

[0104] II.2 Experimental model

[0105] A model of reconstructed human epidermis (RHPE) was constructed using phototype II pigmentation.

[0106] ■ Systemic treatment of tissues with the combination of the invention and Polypodium extracts simulating oral ingestion.

[0107] ■ The tissue is then exposed to a dose of UVA and UVB radiation equivalent to 2 MED (the amount of radiation to which an individual is exposed on average during a day).

[0108] ■ Analysis was performed 4 hours after exposure to UV rays to assess the early acting ability of the combination of the invention.

[0109] II.3 Materials

[0110] 0.5cm 2The EPISKIN Reconstructed Human Pigmentation Phototype II Epidermis (RHPE II, manufactured by EPISKIN SA, 4 Rue Alexander Fleming 69366 Lyon, France) was used for the study. This model reproduces the pigmentation phototype II formed by fully differentiated human keratinocytes and melanocytes after 10 days of culture in air in a chemically defined medium. The batch was tested for the absence of Hepatitis B, Hepatitis C and Mycoplasma. The tissues and culture media were produced according to ISO 9001. Each batch was tested for the absence of HIV, Hepatitis B, Hepatitis C and Mycoplasma.

[0111] The tissues were removed from the agarose nutrient solution under a sterile airflow cabinet immediately after the assay system arrived at the laboratory. The inserts were quickly transferred to a 6-well plate previously filled with maintenance medium and incubated at 37°C, 5% CO2, saturated humidity.

[0112] II.4 Procedure

[0113] For all series, RHPE tissues were tested in triplicate (n=3).

[0114] - NC: untreated and non-irradiated control RHPE in standard medium and harvested after 4 and 24 hours.

[0115] - NC-EtOH 1% - RHPE collected after 4 and 24 hours in medium containing EtOH 1% (negative control)

[0116] -IRR-EtOH 1% - RHPE in medium containing EtOH 1%, irradiated at 2 MED and collected after 4 and 24 hours (positive control)

[0117] - IRR+P1: - RHPE treated with 0.1% P1 (Polypodium cyanum rhizomes) in medium containing 1% EtOH overnight, exposed to 2 MED, and treated 4 and 24 hours after irradiation in medium containing 1% EtOH.

[0118] Irradiation in medium containing 1% EtOH containing product

[0119] - IRR+P3 MIX: - Overnight systemic RHPE treated with mixture P3 (0.1% Polypodium vulgare rhizome (P1) + 0.015% Larix olgensis bark (P2)) in medium containing 1% EtOH, exposed to 2 MED, and treated 4h and 24h after irradiation in medium containing 1% EtOH containing the product.

[0120] To assess the absence of chemical interference, the product (as a systemic exposure) was exposed to killed RHPE for 24 hours, followed by MTT incubation (killed tissues were prepared in water at 37°C over 24 hours).

[0121] Solubility studies: This was done to select a solvent suitable for the product, which was 1% EtOH in culture medium.

[0122] Initial cytotoxicity: The exploration of non-cytotoxic doses for products P1 and P2 was performed by systemic exposure with systemic treatment in medium for 24 hours under agitation throughout the experimental period. Non-cytotoxic doses for efficacy studies were selected after the MTT assay.

[0123] The doses (systemic exposure) containing 1% EtOH for the preliminary MTT test (n=2 tissues for each concentration) were:

[0124] P1: 0.4% / 0.2% / 0.1%

[0125] P2: 0.06% / 0.03% / 0.015%.

[0126] Efficacy Studies: The doses selected for the study were: 0.1% for P1 and 0.015% for P2.

[0127] On the day of tissue arrival, RHPE tissues were incubated under standard culture conditions (37° C., 90% RH, 5% CO 2 ) overnight.

[0128] On the day of the experiment, tissues were treated with systemic exposure overnight in medium containing 0.015% Polypodium vulgare rhizome (unique code = P1) and a mixture of 0.015% Polypodium vulgare rhizome (P1) and 0.1% Larix olgensis bark (P2) (unique code = P3), except for NC (negative control).

[0129] The following day, tissues were irradiated with 2 MED (equivalent to 0.05 J / cm2 UVA+UVB) in PBS using an Oriel 1 KW solar simulator (0.035 mW / cm2) with a xenon arc lamp and irradiance WG320 erythemic filter [mW / cm2].

[0130] After exposure to UV rays, all tissues were transferred to fresh medium containing the product and incubated under standard conditions (37° C., 5% CO 2 , 90% RH) for 4 hours and 24 hours.

[0131] After the incubation period, the culture medium was collected and stored at -20°C for determination of caspase-1 activity. The tissues were gently washed with saline and then each tissue was fixed in formalin for further histological analysis.

[0132] II.5 Inflammasome and caspase-1

[0133] Pathogens, UV rays and stress factors induce a situation of generalized inflammation (inflammasome) at the level of all skin areas, which activates a series of short-term and long-term negative reactions. Regarding this inflammatory cascade, caspase-1 (CASP-1) activation is one of the most important factors: it is directly related to UV-induced oxidative stress and is therefore activated early in response to the stimulus. It leads to the release of proinflammatory cytokines and thus to the inflammatory cascade and subsequent tissue damage.

[0134] It therefore represents an interesting target for preventing and protecting against the appearance of all those cutaneous manifestations caused by excessive and / or cumulative sun exposure and therefore characterized by oxidative stress and inflammation.

[0135] Inflammasomes are intracellular multiprotein complexes that assemble in response to pathogen-associated molecular patterns called PAMPs (pathogen-associated molecular patterns), or various cell or tissue damages called DAMPs (danger-associated molecular patterns) that are capable of inducing inflammatory responses. The processes activated by inflammasomes are not only very important as antimicrobial responses, but also in regulating metabolic pathways and immune responses.

[0136] Inflammasomes originate from the cytosolic compartment of immune and inflammatory cells as an immune response to exogenous or endogenous signals. These complexes arise from the presence of disordered conditions caused by biological, physical, chemical, metabolic agents, high levels of reactive oxygen species (ROS), decreased cytosolic concentrations of potassium ions, and other factors.

[0137] Inflammasomes activate inflammatory responses. They are able to integrate a large number of signals and converge them into a pro-inflammatory response. These multiprotein complexes induce the activation of the inflammatory caspase-1, which in turn activates the cytokines interleukin-1β (IL-1β) and interleukin-18 (IL-18), causing a systemic inflammatory response. In addition, caspase-1 activation can induce a form of inflammatory cell death called pyroptosis.

[0138] II.5.1 Inhibition of Caspase

[0139] method : 1 (Promega, G9951) Inflammasome Assay is a homogenous and bioluminescent method for selectively measuring the activity of caspase-1, a member of the protease (caspase) family that is specific for cysteine ​​aspartate and an important component of the inflammasome. Activation of caspase-1 leads to the processing and release of cytokines IL-1β and IL-18 and pyroptosis (an immunogenic form of cell death). 1 Inflammasome Assays provide a luminescent substrate for caspase-1 in a cleavage reagent optimized for caspase-1 activity and luciferase activity, suitable for assays in cell culture or in media.

[0140] A single addition of the reagent results in cell lysis, cleavage of the substrate by caspase-1, and generation of a stable luminescent signal proportional to caspase activity. Inclusion of the proteasome inhibitor MG-132 in the reagent eliminates proteasome-mediated nonspecific cleavage of the substrate, allowing for sensitive detection of caspase-1 activity. To test the specificity of caspase-1, 1 The assay contains the selective caspase-1 inhibitor Ac-YVAD-CHO. This inhibitor inhibits 99% of caspase-1 activity, but does not substantially inhibit any cross-reacting caspases. Performing the assay in parallel wells with and without Ac-YVAD-CHO allows for specific measurement of caspase-1 activity.

[0141] program: According to the manufacturer's instructions, the collected media were tested. Briefly, equal volumes of culture medium and caspase-1 reaction reagent or caspase-1 reaction reagent containing specific Ac-YVAD-CHO were mixed in two separate 96-well plates for luminescence. After incubation for 120 minutes at RT, TECAN plate reader was used to detect and record luminescence.

[0142] Data collection and analysis: After subtracting the blank (luminescence background) from all collected data, the ALU (arbitrary luminescence unit) of each sample was calculated by subtracting the AFU measured in the presence of the Ac-YVAD-CHO inhibitor from the AFU of the corresponding sample in the absence of the inhibitor. For each experimental treatment, the detection was performed in technical duplicates and biological triplicates n=3.

[0143] result: The combination of the present invention showed a significant reduction (***p<0.001) in the activity of CASP-1, showing an effective early prevention of the inflammatory cascade and thus blocking the inflammasome damage at the skin level (cells from sunstroke, photoaging, oxidative stress, etc.). Figure 1 The results of the tests performed are schematically depicted in a bar graph.

[0144] II.6 Involucrin (protection of the skin barrier structure)

[0145] The stratum corneum represents the most natural physical protection of our skin against the sun and it is essential to maintain its protective function. Involucrin (IVL) is located at the level of keratinocytes through a dense network of disulfide bridges that ensure the impermeability and resistance of the stratum corneum.

[0146] II.6.1 Involucrin

[0147] method :Immunostaining is a histological technique for detecting specific molecules or structures in the cellular compartment of histological sections. The technique is based on the specificity of the antigen-binding antibody used to detect the target molecule and the detection system by fluorescence microscopy or by bright field microscopy if immunochemistry is used using an indirect method. Immunostaining is performed on formalin-fixed and paraffin-embedded (FFPE) sections.

[0148] program : At the end of treatment, tissues were fixed in neutral 10% buffered formalin. After fixation, biological triplicates were included in the same paraffin block and 5 μm sections were obtained.

[0149] The following primary antibodies were used for immunostaining:

[0150] - Anti-Involucrin

[0151] Detection was visualized with Alexa Fluor Plus 555 donkey anti-rabbit, and cell nuclei were counterstained with DAPI solution.

[0152] Data collection and acceptance criteria : Stained sections were visualized with a 3D THUNDER imager microscope, collected with a K5 camera (fluorescence) and processed with LASX 3.7.5 software. For each biological replicate, the entire tissue was visualized with a single solution. For each biological replicate, the entire tissue section was collected with the LASXtilescan technology at a magnification of 20X; in addition, representative images were collected at 40X to be included in the histology report provided separately.

[0153] Protein signals were quantified by evaluating the signal expressed in / on all tissue sections and are shown as total intensity (sum of gray values ​​of all pixels belonging to an object). Statistical analysis (one-way ANOVA with Tukey post hoc test) was performed using Prism 9.

[0154] The analysis was performed in biological triplicate (n=3).

[0155] result: The combination of the invention induces synthesis of involucrin superior to that of irradiated controls, indicating a reinforcing effect on the structure of the barrier function. Figure 2 The results of the tests performed are schematically depicted in a bar graph.

[0156] II.7 Conclusion

[0157] The results of the study confirmed the protective capacity of the combination of the invention. In particular, the following was recorded:

[0158] - Intervention of caspase-1 and, therefore, the inflammasome, provides early protection from potential damage caused by oxidative stress induced by UV exposure and the resulting inflammatory state;

[0159] - It stimulates the synthesis of involucrin, a protein essential for barrier integrity, and therefore proves to protect the skin by strengthening its natural defenses.

Claims

1. A combination comprising or consisting of a dry extract of Polypodium vulgare and a dry extract of larch bark for use in preventing damage from exposure to UVA, UVB, UVC and / or IR rays.

2. The combination for use according to claim 1, wherein the dry extract of Polypodium villosum is a dry extract from the rhizome and / or aerial part of Polypodium villosum.

3. The combination for use according to claim 1 or 2, further comprising at least one active ingredient selected from vitamin C and vitamin B3 or a combination thereof.

4. The combination for use according to any one of claims 1 to 3, in the form of an oral composition, in combination with suitable excipients and / or diluents.

5. Combination for use according to claim 4, wherein the oral composition is a nutraceutical or a medicine or is a food supplement.

6. Combination for use according to any one of claims 4-5, wherein the Polypodium villosum is present in an amount comprised between 100 and 900 mg, preferably between 200 and 700 mg, even more preferably between 300 and 500 mg, and the dry extract of larch bark is present in an amount comprised between 40 and 90 mg, preferably between 50 and 85 mg, even more preferably between 60 and 80 mg.

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