Preparation method of Mastocarpus stellatus extract

Abstract

A method for the preparation of Mastocarpus stellatus extract and cosmetic uses of the extract are disclosed.

Description

[Technical field]

[0001] The present invention relates to a process for preparing a Mastocarpus stellatus extract, the Mastocarpus stellatus extract thus obtained, a cosmetic composition comprising the Mastocarpus stellatus extract, and its use in cosmetics. [Background technology]

[0002] Mastocarpus stellatus, commonly known as carrageenan moss or false Irish moss, is a species in the division Rhodophyceae, a marine algae division of red algae. Mastocarpus stellatus commonly occurs on rocks in the mid and lower intertidal zones. It is commonly found on most coasts of Ireland and the UK. Other recorded locations include France (English Channel, Northeast Atlantic), Iceland, the Faroe Islands, northern Russia to the Rio de Oro, Canada (Newfoundland) to the USA (North Carolina).

[0003] Mastocarpus stellatus grows from a discoid holdfast stem, and the thallus is grooved, unlike the flattened Chondrus crispus. It grows to a height of 10-20 cm and branches dichotomously. The thallus is cartilaginous and reddish-brown, with a greenish or purplish tint. Mature algae show reproductive structures that develop on erect filaments up to 1 mm in diameter. It is reddish-brown, purple, or faint in color.

[0004] According to Michael Gilley, the earliest record of seaweed harvesting in Ireland is attested by a 12th century poem by a monk. An analysis of the Irish seaweed farming market in 2001 found that the estimated annual domestic seaweed harvest of M. stellatus and C. crispus combined was less than 100 tonnes. M. stellatus is harvested during the gametophyte life phase because the later phase, which is high in sulfated carrageenan, is difficult to extract from its rock. The food and pharmaceutical industries are interested in seaweeds that have antioxidant, anticoagulant, thickening or gelling properties. In addition to its health properties and applications, the gelling properties of M. stellatus allow it to make biodegradable films. This could be a sustainable and edible alternative to plastics in food preservation and the development of functional foods.

[0005] EP 1 743 628 A1 relates to a cosmetic composition comprising a red algae extract containing a carrier and a combination of 25-50% by weight of floridoside and 10-25% by weight of isethionic acid in a mass ratio of 1:5. This composition can be used to hydrate the skin and / or prevent skin aging.

[0006] FR 2 946 878 B1 relates to the cosmetic use of floridoside or red algae extracts as melanin production inhibitors. Summary of the Invention [Problem to be solved by the invention]

[0007] Surprisingly, it has now been found that by using a special extraction method, it is possible to obtain Mastocarpus stellatus extract that has fat-dissolving activity and can increase skin elasticity, thus reducing the appearance of a double chin. [Means for solving the problem]

[0008] Thus, in a first aspect, the present invention provides a method for preparing a Mastocarpus stellatus extract, the method comprising the steps of: (i) extracting Mastocarpus stellatus with water to obtain a first liquid phase and a first solid phase; (ii) acidic hydrolysis of the first solid phase at elevated temperature; (iii) phase separating to obtain a second liquid phase and a second solid phase; and (iv) combining the first and second liquid phases to obtain a Mastocarpus stellatus extract.

[0009] In a second aspect, the present invention relates to a Mastocarpus stellatus extract obtained by the method of the present invention or obtainable by the method of the present invention.

[0010] In a third aspect, the present invention relates to a cosmetic composition comprising the Mastocarpus stellatus extract of the present invention and a cosmetically acceptable excipient.

[0011] In a fourth aspect, the present invention relates to a method for reducing a double chin comprising the step of topically applying to the double chin a Mastocarpus stellatus extract, in particular a Mastocarpus stellatus extract of the present invention or a cosmetic composition of the present invention.

[0012] In a fifth aspect, the present invention relates to the use of the Mastocarpus stellatus extract of the present invention or the cosmetic composition of the present invention for reducing a double chin, for enhancing lipolysis, for increasing skin elasticity, for stimulating collagen synthesis, for hydrating the skin, for reducing the effects of skin ageing and / or for preventing the effects of skin ageing. [Brief description of the drawings]

[0013] [Figure 1] FIG. 1 shows the AFM measurements. [Diagram 2] FIG. 2 shows the distribution of collagen fiber orientation in two dimensions as analyzed photographically. [Diagram 3] FIG. 3 shows exemplary photographs of AEVA HE® measurements on one volunteer who applied a cream containing 1% Mastocarpus stellatus extract. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] The invention and its preferred features are described in more detail below. For the avoidance of doubt, preferences, options, specific features and similarities set forth with respect to given aspects, features and parameters of the invention are to be considered as being disclosed in combination with any or all other preferences, options, specific features and similarities as set forth with respect to the same or other aspects, features and parameters of the invention, unless the context indicates otherwise.

[0015] The method of the present invention includes aqueous extracting Mastocarpus stellatus to obtain a first liquid phase and a first solid phase.

[0016] Water extraction can be performed on the entire Mastocarpus stellatus plant, or alternatively, only certain parts of the plant, such as the stem and / or thallus, can be used.

[0017] Prior to water extraction, Mastocarpus stellatus can be dried and / or comminuted, for example by cutting or grinding or squeezing or mincing, or any other suitable method known to those skilled in the art.

[0018] Thus, in one embodiment, Mastocarpus stellatus is dried prior to water extraction. The algae can be dried in any suitable manner, such as in an oven at low temperature (such as about 40°C) or in the sun. Using dried algae allows for easier storage and logistics. Alternatively, it is also possible to use, for example, fresh or frozen algae.

[0019] In one embodiment, Mastocarpus stellatus is processed into smaller pieces, in particular cut and / or crushed, prior to aqueous extraction. This improves the efficiency of the extraction process. Preferably, Mastocarpus stellatus is processed into pieces of about 15-20 mm in length prior to aqueous extraction (e.g., the pieces may have the following size distribution: 5-10% above 2360 μm, 80-90% below 2360 μm and above 1000 μm, 10-15% below 1000 μm). This allows the extraction process to be optimized while avoiding the need to extract carrageenan in a first extraction step. In the method of the present invention, preferably, all carrageenan contained in the algae is subjected to acid hydrolysis.

[0020] Aqueous extraction is typically carried out using water, especially deionized water, but alternatively, for example, regular tap water or water containing additives such as glycerol, propanediol, ethanol, other polar solvents, or buffers can be used.

[0021] In one embodiment, the water extraction involves macerating Mastocarpus stellatus in water, particularly deionized water.

[0022] Water extraction, particularly maceration in water, can be carried out at a suitable concentration of Mastocarpus stellatus in water.

[0023] In one embodiment, the water extract comprises macerating Mastocarpus stellatus in water, particularly deionized water, at a concentration of about 2-10%, more preferably about 4-5%, for example about 4.8%.

[0024] Throughout this disclosure, the indications of percentage (%) refer to percentage by weight (w / w) unless otherwise specified.

[0025] Water extraction, especially maceration in water, can be carried out at room temperature, alternatively, slightly higher or lower temperatures can be used.

[0026] In one embodiment, the aqueous extract comprises macerating Mastocarpus stellatus at a temperature of about 10-30°C, more preferably about 20°C.

[0027] The water extraction, particularly the maceration in water, can be carried out for a suitable period of time, depending on the temperature and / or concentration used.

[0028] In one embodiment, the water extract comprises macerating Mastocarpus stellatus for about 5 to 60 minutes, more preferably for about 15 minutes.

[0029] In a particular embodiment, the water extract comprises macerating Mastocarpus stellae in water, particularly deionized water, at a concentration of about 2-10%, more preferably about 4-5%, at a temperature of about 10-30°C, more preferably about 20°C, for about 5-60 minutes, more preferably about 15 minutes.

[0030] The first liquid phase and the first solid phase can be separated using any suitable method. The preferred method may vary, for example, depending on the scale of the water extraction, but also on other factors such as particle size, viscosity of the liquid phase, or available equipment. Centrifugation, sieving, and / or filtration are particularly suitable methods.

[0031] In one embodiment, the first liquid phase and the first solid phase are separated by one or more of centrifugation, sieving, and filtration.

[0032] After the first phase separation, the first liquid phase may be stored under suitable conditions. For example, the first liquid phase may be stored at ambient temperature protected from light for several hours (e.g., about 3-4 hours) during the remainder of the extraction process. Additionally, the first liquid phase may be stabilized by adding a suitable stabilizer, such as a (polar) solvent. For example, the first liquid phase may be stabilized by adding 20% ​​propanediol.

[0033] The acid hydrolysis of the first solid phase is used specifically to hydrolyze the carrageenan contained in the algae material.

[0034] The acid hydrolysis of the first solid phase may be carried out at any suitable pH. Depending on the degree of hydrolysis desired, a slightly higher or lower pH can be selected.

[0035] In one embodiment, the acid hydrolysis is carried out at a pH of about 1-4, more preferably at a pH of about 1.5-2.5, and most preferably at a pH of about 2.

[0036] The desired pH can be obtained by adding a suitable acid, with sulfuric acid, hydrochloric acid, citric acid and mixtures thereof being particularly suitable.

[0037] In one embodiment, the acid hydrolysis comprises adding sulfuric acid, hydrochloric acid, citric acid or any mixture thereof.

[0038] The ratio of Mastocarpus stellatus to acid can also be adjusted depending on the desired degree of hydrolysis. A suitable ratio is, for example, about 3-6% sulfuric acid, preferably about 4.5% sulfuric acid.

[0039] The acid hydrolysis is carried out at elevated temperatures. Higher or lower temperatures can be selected depending on the degree of hydrolysis desired. In one embodiment, the acid hydrolysis is carried out at a temperature of about 50-90°C, more preferably at a temperature of about 70-90°C, and most preferably at a temperature of about 80°C.

[0040] The duration of the acid hydrolysis can also be adjusted depending on the desired degree of hydrolysis: the longer the duration, the lower the degree of polymerization.

[0041] In one embodiment, the acid hydrolysis is carried out for a period of about 1 hour to 3 hours, more preferably about 2 hours.

[0042] Acid hydrolysis can be stopped, for example, by increasing the pH or decreasing the temperature.

[0043] In one aspect, the process of the invention further comprises the step of adjusting the pH after acid hydrolysis to a pH of about 4 to 8, more preferably to a pH of about 4.5 to 6, especially a pH of about 5.

[0044] The pH can be adjusted by adding a suitable base, for example a mineral base such as sodium hydroxide.

[0045] After acid hydrolysis, and optionally pH adjustment, phase separation is performed to obtain a second liquid phase and a second solid phase, which can be achieved by any suitable means, such as centrifugation, filtration, vibrating sieving, and / or wringing.

[0046] Thus, in one aspect, the phase separation in step (iii) comprises one or more of centrifugation, filtration, vibrating sieving, and squeezing.

[0047] Optionally, a suitable stabilizer may be added to the second liquid phase prior to combination with the first liquid phase. For example, the second liquid phase may be stabilized by adding a (polar) solvent, for example 20% propanediol. Preferably, the same stabilizer is used for both the first and second liquid phases.

[0048] Finally, the first liquid phase and the second liquid phase are combined to obtain the Mastocarpus stellatus extract of the present invention.

[0049] Optionally, the Mastocarpus stellatus extract of the present invention may be filtered, for example to 0.3 μm, after combining the first and second liquid phases.

[0050] Optionally, the Mastocarpus stellatus extract of the present invention may be heated, for example to about 60° C., after combining the first and second liquid phases, which may reduce the pink color of the extract, possibly by destroying pigments such as phycoerythrin.

[0051] The following table shows a comparison of the first liquid phase, the second liquid phase, and the Mastocarpus stellatus extract of the present invention. [Table 1]

[0052] Mastocarpus stellatus extract can be used as is, diluted or concentrated depending on the intended use and desired concentration.

[0053] The Mastocarpus stellatus extract of the present invention may be stored under suitable conditions, for example at ambient temperature, and may preferably be protected from light.

[0054] The present invention also relates to a Mastocarpus stellatus extract obtained by the method of the present invention or obtainable by the method of the present invention.

[0055] As will be understood by one of skill in the art, the term "obtainable from" as used herein means that the extract may be obtained or isolated from the plant, or may be obtained from an alternative source, for example by chemical synthesis or enzymatic production, whereas the term "obtained" as used herein means that the extract is obtained directly from the plant source.

[0056] The Mastocarpus stellatus extract of the present invention has been found to have several beneficial cosmetic effects, which have been demonstrated in both in vitro and clinical studies (see examples below).

[0057] In particular, it has been found that Mastocarpus stellatus extract can reduce double chin, promote lipolysis, increase skin elasticity, stimulate collagen synthesis, hydrate the skin, reduce the effects of skin aging, and prevent the effects of skin aging.

[0058] Mastocarpus stellatus extract is advantageously applied topically to the skin.

[0059] In a further aspect, the present invention provides a cosmetic composition comprising the Mastocarpus stellatus extract of the present invention and a cosmetically acceptable excipient. The cosmetic composition of the present invention is intended for topical application.

[0060] The concentration of Mastocarpus stellatus extract in the cosmetic composition should be selected to obtain the desired effect.

[0061] Any excipient commonly used in the preparation of cosmetic preparations for use on human skin can be used in the present invention. Suitable excipients include, but are not limited to, ingredients that can affect the sensory properties, skin penetration, and bioavailability of the Mastocarpus stellatus extract. More specifically, they include liquids such as water, oils, or surfactants, which may be of petroleum, animal, vegetable, or synthetic origin, including, but not limited to, peanut oil, soybean oil, mineral oil, sesame oil, castor oil, polysorbates, sorbitan esters, ether sulfates, sulfates, betaines, glycosides, maltosides, fatty alcohols, nonoxynol, poloxamer, polyoxyethylene, polyethylene glycol, dextrose, glycerol, digitonin, and the like.

[0062] Preparations for topical application to the skin may take any physical form. Illustratively, cosmetic compositions, and in particular skin care compositions, may be in the form of liposomal compositions, mixed liposomes, oleosomes, niosomes, ethosomes, milliparticles, microparticles, nanoparticles and solid-lipid nanoparticles, vesicles, micelles, surfactant mixed micelles, surfactant-phospholipid mixed micelles, millispheres, microspheres and nanospheres, lipospheres, millicapsules, microcapsules and nanocapsules, as well as microemulsions and nanoemulsions, which may be added to achieve a higher penetration of the Mastocarpus stellatus extract.

[0063] Cosmetic compositions, and particularly skin care compositions, can be prepared in any solid, liquid, or semi-solid form useful for topical or transdermal application to the skin. Thus, these preparations for topical or transdermal application include, but are not limited to, creams, numerous emulsions, such as, but not limited to, oil-in-water and / or silicone emulsions, water-in-oil and / or silicone emulsions, water / oil / water or water / silicone / water emulsions, and oil / water / oil or silicone / water / silicone emulsions, micro-emulsions, emulsions and / or solutions, liquid crystals, anhydrous compositions, aqueous dispersions, oils, milks, balsams, foams, aqueous or oily lotions, aqueous or oily gels, creams, hydro-alcoholic solutions, hydro-glycolic solutions, hydrogels, liniments, sera, soaps, face masks, serums, polysaccharide films, ointments, mousses, pomades, pastes, powders, bars, pencils, and sprays or aerosols (sprays), including leave-on and rinse-off formulations.

[0064] Thus, the present invention also provides a skin care composition.

[0065] The cosmetic composition of the present invention may further comprise other cosmetic active agents, such as anti-aging agents, moisturizing agents or hydrating agents.

[0066] In one embodiment, the cosmetic composition of the present invention further comprises an anti-aging agent comprising a mixture of mannose-6-phosphate and mannose, as described in WO 2020 / 201185. The disclosure of WO 2020 / 201185 regarding cosmetic active agents and embodiments and uses thereof is incorporated herein by reference.

[0067] In yet another aspect, the present invention also provides a method for reducing a double chin comprising topically applying a Mastocarpus stellatus extract, in particular a Mastocarpus stellatus extract according to the present invention, or a cosmetic composition according to the present invention to the double chin.

[0068] In a further aspect, the present invention also relates to the use of the Mastocarpus stellatus extract of the present invention or the cosmetic composition of the present invention for reducing a double chin.

[0069] In a further aspect, the present invention also relates to the use of a Mastocarpus stellatus extract according to the invention or a cosmetic composition according to the invention for promoting lipolysis.

[0070] In a further aspect, the present invention also relates to the use of the Mastocarpus stellatus extract of the present invention or the cosmetic composition of the present invention for increasing skin elasticity.

[0071] In a further aspect, the present invention also relates to the use of the Mastocarpus stellatus extract of the present invention or the cosmetic composition of the present invention for stimulating collagen synthesis.

[0072] In a further aspect, the present invention also relates to the use of the Mastocarpus stellatus extract of the present invention or the cosmetic composition of the present invention for hydrating the skin.

[0073] In a further aspect, the present invention also relates to the use of the Mastocarpus stellatus extract of the present invention or the cosmetic composition of the present invention for reducing the effects of skin ageing.

[0074] In a further aspect, the present invention also relates to the use of the Mastocarpus stellatus extract according to the invention or the cosmetic composition according to the invention for preventing the effects of skin ageing. EXAMPLES

[0075] The present invention is further illustrated by the following non-limiting examples:

[0076] Example 1: Mastocarpus stellatus extract A suspension of Mastocarpus stellatus at 4.8% dry matter in water was prepared (20 g dry algae + 396.67 g percolation water). The algal material was first cut with scissors into small pieces of 9 mm length and then macerated with stirring for 15 min at room temperature (22 °C). The biomass (= first solid phase) and the supernatant (= first liquid phase) were separated by filtration (Whatman filter, 40 µm). 1,3-propanediol was added to the supernatant at a concentration of 20% of the total volume, obtaining extract 1. This was then stored at room temperature and protected from light for approximately 3-4 h while extract 2 was prepared.

[0077] The insoluble part of the previous extraction (=first solid phase) was resuspended at 4.8% of the dry matter in percolation water. The suspension was then stirred and acidified to pH = 2 with 96% sulfuric acid solution and then heated to 80°C. The acid hydrolysis was continued for 2 hours at 80°C. After 2 hours, the suspension was cooled and neutralized to pH = 5 with 10 M sodium hydroxide solution. The extract was centrifuged (4500 rpm, 20°C, 20 min), the supernatant was separated from the solids and 1,3-propanediol was added to the supernatant (=second liquid phase) at a concentration of 20% of the total volume to obtain extract 2.

[0078] Finally, the two extracts 1 and 2 were combined, stirred and heated to 60° C. After heating for 30 minutes, the mixture was immediately filtered successively through 2.5 μm and 0.3 μm to obtain the Mastocarpus stellatus extract according to the present invention.

[0079] The extract thus obtained had a pH of about 5.45, a dry mass content of about 1.63%, a Gardner colour of about 2.1% and a floridoside content of about 0.86 g / l.

[0080] Example 2: Transcriptome analysis of human fibroblasts Normal human dermal fibroblasts (NHDFs) were seeded at 300,000 cells per well in 6-well plates. After 48 h of culture in Dulbecco's modified Eagle's medium supplemented with 10% FCS, NHDFs were rinsed twice with phosphate-buffered saline (PBS) and left overnight in FCS-free medium before stimulation. Cells were stimulated with Mastocarpus stellatus extract at 0.5% and compared to the untreated condition. After 6 and 18 h of stimulation, total RNA was extracted by the extractol method (Rio DC, Ares M Jr, Hannon GJ, Nilsen TW. Purification of RNA using TRIzol (TRI Reagent). Cold Spring Harb Protoc. 2010 Jun;2010(6):pdb.prot5439. doi: 10.1101 / pdb.prot5439.PMID:20516177). RNA quality was controlled and reverse transcription was performed to obtain cDNA. RT-qPCR was performed on specific plates designed to study the transcriptomic expression of different genes involved in skin elasticity in NHDFs, using 10 ng of cDNA per well. Gene expression results obtained in fibroblasts were normalized according to the PES1 (pescadilloribosomal biogenesis factor 1), GADD45A (growth arrest and DNA damage inducible alpha), and HMBS (hydroxymethylbilane synthase) housekeeping genes. Data are expressed as fold change relative to the untreated condition.

[0081] result To investigate whether Mastocarpus stellatus extract can improve skin elasticity, a transcriptomics study was performed on fibroblasts treated with Mastocarpus stellatus extract at 0.5% to analyze the expression of various genes involved in skin elasticity.

[0082] After 6 hours of treatment, Mastocarpus stellatus extract significantly increased the expression of genes involved in elastic fiber organization such as FBLN5, LOXL1, and MFAP2 by +34%, +32%, and +10%, respectively. It also decreased the expression of HPSE, a gene that codes for a protein involved in the degradation of elastic fibers, by -53%.

[0083] Interestingly, even after 18 h of treatment with Mastocarpus stellatus extract, the expression of FBLN5 and MFAP2 was significantly increased by + 24% and + 24%, respectively, while the expression of HPSE was decreased by − 40%.

[0084] Mastocarpus stellatus extract also stimulated the expression of genes involved in extracellular matrix structure, such as COL3A1 and CD44, by +33% and +58%, respectively.

[0085] The results are summarized in the table below: [Table 2]

[0086] This transcriptomics study identified Mastocarpus stellatus extract as a promising candidate for improving skin elasticity.

[0087] Example 3: Proteomic analysis of human skin explants The effect of Mastocarpus stellatus extract on skin elasticity was further evaluated at the proteomic level.

[0088] Sample preparation Skin explants from young donors (28 years old) and mature donors (59 years old) maintained viability at the air-liquid interface. Skin explants from young donors were left untreated, whereas skin explants from mature donors were treated topically with Mastocarpus stellatus extract at 1% and compared to the untreated condition. Treatment and medium (MIL217C from Biopredic International) were renewed daily for 5 days. After this 5-day stimulation, skin explants were rinsed twice with PBS and frozen at -80°C.

[0089] Sample processing The sheared tissue was added to the zirconia oxide bead mix and 700 μl of iST LYSE buffer (deoxycholic acid, TCEP, chloroacetamide). Samples were lysed by two bead-beating cycles. The homogenate was transferred to a Diagenode protein extraction tube and co-extracted nucleic acids and organelles were sheared by microcavitation (Bioruptor Pico, Diagenode). Proteins were solubilized, reduced and alkylated by boiling in iST LYSE buffer (deoxycholic acid, TCEP, chloroacetamide). Protein concentration was measured by the BCA method (Walker JM. The bicinchoninic acid (BCA) assay for protein quantification. Methods Mol Biol. 1994;32:5-8. doi: 10.1385 / 0-89603-268-X:5. PMID:7951748.). Peptide extracts were prepared according to the iTS method (Stage Chip). 50 μg of protein was digested with a mixture of LysC and trypsin. Peptides were purified by mixed-mode reversed-phase cation exchange SPE (solid-phase extraction, PreOmics GmbH), dried, and solubilized in 100 μl of 3% acetonitrile-0.1% formic acid in water. Peptide concentrations were measured using the BCA method.

[0090] LC-MS / MS 300ng of peptides were injected in triplicate for each sample. Chromatography was performed on an Ultimate 3000 (Dionex) instrument using a C18 (75μm x 50 cm, 2μm material) column with a 3 min trapping step on the precolumn followed by a gradient of 2.5% to 35% acetonitrile over 120 min at a flow rate of 300nl / min. Data were acquired using a Q-Exactive (Thermo) mass spectrometer. MS scans were performed at a resolution of 70,000 and an accumulation time of 60 ms. MS / MS scans were performed at a resolution of 17'500 on the 10 most intense ions in each cycle with an accumulation time of 60 ms. 6545 cycles were performed, resulting in an average of 17 cycles per chromatographic peak.

[0091] Protein identification Proteins were identified using the SEQUEST-HT algorithm (Tabb DL.SEQUEST Family Tree.J Am Soc Mass Spectrom 2015;26(11):1814-1819. doi:10.1007 / s13361-015-1201-3) against a database of human reference proteomes mined from Uniprot and enzymes used for digestion. Search parameters were enzyme = trypsin (complete), allowed misclearance = 2, precursor error tolerance = 10 ppm, fragment error tolerance = 0.02 Da, dynamic modification = oxidation (M), deamidation (N / Q), protein terminal modification = acetylation, static modification = carbamidomethyl (C).

[0092] False discovery rate (FDR) determinations were performed using the Percolator algorithm (Spivak M, Weston J, Bottou L, Kall L, Noble WS. Improvements to the Percolator algorithm for peptide identification from shotgun proteomics datasets. J. Proteome Res. 2009 July 8 (7): 3737-45. doi: 10.1021 / pr801109k. PMID: 19385687; PMCID: PMC2710313.).

[0093] All spectra reported by SEQUEST-HT with less than high confidence (and therefore considered not identified) were reprocessed by the MS Amanda 2.0 algorithm against the same database as above. Search parameters were: enzyme = trypsin (complete), allowed misclearance = 2, precursor error tolerance = 10 ppm, fragment error tolerance = 0.02 Da, dynamic modifications = oxidation (M) (P), deamidation (N / Q), protein terminal modifications = acetylation, Met loss, Met loss + acetyl, static modifications = carbamidomethyl (C). False discovery rate (FDR) determination was performed using the Percolator algorithm.

[0094] Protein quantification Data were processed using Minora and Feature Mapper in Proteome Discoverer 2.3 software (Thermo Fisher Scientific). Peak integration parameters were: post-acquisition recalibration = True (fine parameters), minimum trace length = 5, minimum number of isotopes = 2, maximum delta RT for isotopes = 0.2 min, PSM (peptide-spectral match) confidence of integration = high.

[0095] Chromatographic alignment parameters were: RT alignment = TRUE, parameter tuning = fine, maximum RT shift = 5 min, mass tolerance 10 ppm. Feature mapping parameters were: RT tolerance = auto, mass tolerance = auto, S / N threshold = 2.

[0096] Statistical analysis was performed using the precursor ion quantifier node of Proteome Discoverer 2.4 software (Thermo Fisher Scientific).

[0097] Common quantification settings were: Peptides to use = Unique + RAZOR (Unique = peptides not shared by different proteins or protein groups, RAZOR = peptides shared by multiple protein groups, but only used to quantitate proteins with the highest number of unique peptides and longest amino acid sequences), Consider protein groups for peptide uniqueness = True, Reject Quan results with missing channels = False. Precursor quantification settings were: Precursor abundance based on area, Minimum number of replicates feature = 50% (a peptide must be detected in at least 50% of samples from one group to be used for quantification). Normalization settings: Total peptide abundance (Calculate the sum of abundance values ​​in each injection for all identified peptides. The injection with the highest total abundance is used as the reference and abundance values ​​in all other injections are corrected by a constant factor for each injection. This ensures that the total abundance is ultimately the same for all injections.)

[0098] Data Selection The first selection was performed by selecting only proteins significantly affected by aging (mature vs. young donors) and then restoring them with Mastocarpus stellatus extract (treated vs. untreated mature donors). This protein selection identified proteins involved in the structure and elasticity of the skin dermis, and five proteins were highlighted.

[0099] result First, we assessed the impact of skin aging on the proteome by comparing young (28 years old) and mature (59 years old) donors. We found a significant decrease in the expression of proteins involved in elastic fiber organization such as MFAP4, TIMP1, and FBLN5 (-10%, -60%, and -76%, respectively), as well as other structural proteins of the dermis such as COL1A1 and COL2A1 (-75%, respectively), confirming the detrimental effect of skin aging on dermal structure.

[0100] The results compared with younger donors are summarized in the table below. [Table 3]

[0101] We next investigated the effect of Mastocarpus stellatus extract on mature donors, whose dermal structure changes with age. The expression of the above five proteins, which was decreased compared to young donors, was found to be significantly increased after 5 days of treatment with Mastocarpus stellatus extract. Indeed, with regard to elastic tissue organization, the expression of MFAP4, TIMP1, and FBLN5 was increased by + 64%, + 23%, and + 95%, respectively. Similarly, the use of Mastocarpus stellatus extract increased the expression of COL1A1 and COL2A1 by + 17% and + 14%, respectively.

[0102] The results compared with mature donors are summarized in the table below. [Table 4]

[0103] These results confirm at the protein level that Mastocarpus stellatus extract is beneficial for the structure and organization of elastic fibers in the dermis.

[0104] Example 4 Analysis of skin biomechanical properties Sample preparation Skin explants from young donors (19 years old) and mature donors (49 years old) maintained viability at the air-liquid interface. Skin explants from young donors were left untreated, whereas skin explants from mature donors were treated topically with Mastocarpus stellatus extract at 1% and compared to the untreated condition. Treatment and medium were renewed daily for 3 days. After this 3-day stimulation, skin explants were rinsed twice with PBS and cryopreserved in OCT™ compound mounting medium and cryosectioned at 20 μm thickness.

[0105] Young's modulus analysis by atomic force microscope (AFM) The atomic force microscope used in this study is a Bioscope Resolve (Bruker) with an additional epifluorescence microscope (Leica DMi8). This setup allows the AFM probe to be precisely positioned on the sample. This unique combination also allows for correlative imaging from mechanical to fluorescent acquisition.

[0106] In this study, QNM (Quantitative Nanomechanical Mapping) Peakforce® mode was used. The AFM probe carries a theoretical spring constant of 0.4 N / m, a radius of curvature less than 10 nm. Before each use, the deflection sensitivity of the probe was measured on sapphire and its spring constant was also calibrated with the thermal noise method (Kim Y, Mandriota N, Goodnight D, Sahin O. Calibration of T-shaped atomic force microscope cantilevers using the thermal noise method. Rev Sci Instrum. 2020 Aug 1;91(8):083703. doi: 10.1063 / 5.0013091. PMID: 32872926; PMCID: PMC7413748.). Force measurements were performed in air (PBS 1X, batch number: CP20-3404).

[0107] The AFM measurement consists in obtaining a force volume applied to the dermis, as shown in Figure 1. Each pixel in the image corresponds to a force indentation curve from which the elastic modulus (Ea) is extracted. Some anomalous data are suppressed by the software, so the number of values ​​may vary depending on the area.

[0108] Collagen fiber organization analysis by biphotonic microscopy A ZEISS LSM880 inverted confocal microscope was used for SHG (second harmonic generation) imaging. The laser used was a coherent two-photon pulsed Chameleon laser. The objective was a 40x water objective "C-Apochromat". To image collagen, samples were excited with a wavelength of 900 nm and light was collected with a 445 nm filter. Three large images of 600 μm x 600 μm size were created for each condition.

[0109] Data analysis was performed using Image J and analysis of the mean grey value on three surfaces of 50 x 50 μm size per image.

[0110] Collagen fiber orientation studies were performed using Rozeta software in three regions of 50 x 50 μm per image.

[0111] Result: Restoration of skin elasticity Elastic modulus measurements demonstrated the effect of skin aging on both the epidermis and dermis: in fact, the elastic modulus of the 49-year-old donor increased significantly and strongly compared to the 19-year-old donor, meaning that the AFM probe required a higher force to deform mature skin compared to younger skin, and that skin loses elasticity with age.

[0112] However, application of Mastocarpus stellatus extract to the surface of mature skin demonstrated a significant reduction in the elastic modulus of the epidermis and dermis, improving skin suppleness. The results are summarized in the table below: [Table 5]

[0113] Results: Recovery of the dermal scaffold The suppleness / stiffness properties of skin are primarily induced by the organization of the dermal scaffold. To understand how elastic modulus is affected by aging, we investigated the organization of collagen fibers in the dermis.

[0114] Collagen fibers from young donors were found to exhibit a two-dimensional organization, meaning that the fibers were oriented in at least two directions, forming a scaffold. On the other hand, fibers in the dermis of mature donors showed a more isotropic organization, meaning that the fibers were oriented mainly in one direction, meaning that the scaffolding of the dermis was lost. Changes in the orientation of collagen fibers with ageing could lead to a loss of scaffolding and directly translate to a loss of skin suppleness.

[0115] Interestingly, topical application of Mastocarpus stellatus extract on mature donor skin explants led to a reorientation of collagen fibers in a 2D organization. This 2D organization favors the reorganization of the dermal scaffold and could explain the improved biomechanical properties of the skin shown by AFM. Figure 2 shows the distribution of collagen fiber orientation in 2 dimensions analyzed photographically. For each direction, the longer the bar, the higher the number of fibers oriented in the respective direction.

[0116] Example 5: lipolytic activity Another factor involved in the formation of a double chin is the accumulation of adipose tissue in the chin. To determine whether Mastocarpus stellatus extract could reduce the volume of the double chin by reducing the content of adipose tissue, mature adipocytes were embedded in a 3D matrix.

[0117] 3D culture of human mature adipocytes Mature adipocytes were obtained from subcutaneous adipose tissue of a 30-year-old woman with a body mass index (BMI) of 28.7 kg / m2, and the tissue was given upon informed consent. Mature adipocytes were isolated from the subcutaneous adipose tissue after digestion with collagenase. The isolated adipocytes were washed with washing buffer and encapsulated in peptide hydrogels to form 3D adipocyte capsules with a size of 25 μl. The formation of adipocyte capsules followed an internal standardized protocol and had approximately equal numbers of adipocytes between capsules. The cells were then incubated at 37 °C for 24 h to stabilize. Treatment with 0.5% Mastocarpus stellatus extract was initiated at D0, changing the medium. The culture medium was replaced daily up to 4 days of culture. After each culture medium change, cell secretions were collected, centrifuged, and frozen at -80 °C for further analysis. Each culture condition was performed in triplicate.

[0118] Quantification of extracellular secretion of glycerol and adiponectin Culture media after 72 h of treatment were collected. Adiponectin and glycerol concentrations were assessed by ELISA and colorimetric assays, respectively, according to the specific kits (Adiponectin kit, Duoset, DY1065, R&D Systems; Glycerol kit, Randox, GY105) as per the manufacturer's recommendations.

[0119] Results: Glycerol release Evaluation of glycerol released from adipocytes was performed in adipocyte culture medium after 72 hours of treatment. Isoproterenol at 1 μM (positive control) significantly increased glycerol release by +196% due to its lipolytic effect on adipocytes, corroborating the model. Mastocarpus stellatus extract at 0.5% also had a significant effect, releasing +36% glycerol after 72 hours of treatment, proving its lipolytic activity.

[0120] The results are summarized in the table below: [Table 6]

[0121] Results: Adiponectin secretion Adiponectin is another factor secreted by mature adipocytes. After 72 hours of treatment, 0.5% Mastocarpus stellatus extract was found to significantly increase adiponectin secretion by +48%, confirming its lytic activity on adipose tissue.

[0122] The results are summarized in the table below: [Table 7]

[0123] Example 6 Clinical studies - Skin biomechanical properties, double chin reduction, V-shape improvement Prescription In the first set of clinical studies described below, cosmetic preparations having the following INCI formulas were used:

[0124] AQUA / WATER, CETYL ALCOHOL, GLYCERYL STEARATE, PEG-75 STEARATE, CETETH-20, STEARETH-20, ISODECYL NEOPENTANOATE, MASTOCARPUS STELLATUS EXTRACT, GLYCERIN, PHENOXYETHANOL, DIMETHICONE, PHENOXYETHANOL, METHYLPARABEN, PROPYLPARABEN, ETHYLPARABEN, FRAGRANCE

[0125] In the placebo composition, the Mastocarpus stellatus extract was omitted. More specifically, [Table 8]

[0126] Panel Description A single-center study was conducted on 44 volunteers with dry skin (corneometry measurements <70 au), crow's feet, and chin ptosis. The volunteers were divided into two groups of 22 volunteers each: - Group 1: 22 women with an average age of 49 ± 6 years testing a cream containing Mastocarpus stellatus extract - Group 2: 22 women with a mean age of 49 ± 5 years who were tested on a placebo cream

[0127] The study was carried out in accordance with Bio EC standard operating procedures and in compliance with the guidelines of the Scientific Committee for Consumer Safety (SCCS) and regulations established in the “Guia para investigaciones con seres humanos” (Guidelines for research involving humans).

[0128] Volunteers applied a cream containing 1% Mastocarpus stellatus extract or a placebo cream, respectively, in the morning and evening every day for 28 days.

[0129] In this study, biomechanical properties were analyzed using Cutometery® analysis and fringe protection with AEVA HE®, as described below.

[0130] Measuring the Biomechanical Properties of Skin with Cutometer® Measuring the mechanical properties of the skin allows the assessment of the functional state of elastic tissue structures (elastic fibers, curvature of connective bundles, wrinkling of the stratum corneum) and viscous-behaving tissue structures (interstitial fluid, internal deposits).

[0131] The study was carried out using a Cutometer® MPA 580 from Courage & Khazaka. The measurement principle is based on the suction method. A negative pressure is generated inside the device, which draws the skin into the cylindrical opening (2 mm diameter) of the probe. Inside the probe, the penetration depth is determined by an optical measurement system. Each suction phase is followed by a relaxation phase.

[0132] The following programs were used in this study: - Cycle length: 4 seconds (suction: 2 seconds, relaxation: 2 seconds) - Negative pressure: 450 mbar - Chamber diameter: 2 mm - Measurement area: fine lines around the eyes

[0133] The resistance of the skin to negative pressure and its ability to return to its original position were plotted as curves at the end of each measurement, from which parameters could be calculated. - During the suction phase, the deformation of the skin due to the negative pressure determines first the elastic resistance and then the viscous component, which together describe the skin's hardness. - During the relaxation phase, the immediate recovery of the skin determines the skin elasticity, while the delayed return of the skin to its initial position measures the viscoelastic component.

[0134] The study focused on the following parameters: - R0 or Uf, which describes the amplitude of the skin during the suction phase: at the same pressure, the softer the skin, the higher the amplitude. This parameter therefore evaluates the non-essentiality of the viscoelasticity, i.e. the stiffness of the skin. - R5 represents the net elasticity (Ur / Ue): the elastic part of the relaxed region (Ur) divided by the elastic part of the attracted region (Ue).

[0135] These parameters were measured on D0 and D28.

[0136] AEVA HE® Analysis: Focus on Double Chin Volume (V-Shape Reshaping) Based on a patented (US 7,821,649) fringe projection unit combined with stereo imaging technology, the AEVA-HE® system is capable of a variety of measurements, from wrinkle reduction to body reshaping. It is designed to quantify the effectiveness of cosmetic, aesthetic and dermatological products and treatments. In particular, it can be used to assess facial sagging and double chin.

[0137] statistical analysis For in vivo studies, the Shapiro Wilk test was used to verify whether the raw data followed the Gaussian law. For normally distributed data, either the unpaired or paired Student's t test was used to compare means. For non-normally distributed data, the Wilcoxon test (paired) was used for paired data, and the Kruskal-Wallis test followed by the Mann-Whitney U test (unpaired) was used for unpaired data, respectively. Whatever statistical test was used, results were deemed significant as follows: # p<0.1, * p<0.05, ** p<0.01, *** p<0.001. For the analysis of the results of the self-assessment questionnaire and the daily log, chi-square tests were made (dichotomous analysis consisting of comparing the number of relevant answers).

[0138] The result: increased skin firmness and elasticity In the first clinical study, the dermal biomechanical properties of skin were measured after application of a cream containing 1% Mastocarpus stellatus extract™ or a placebo cream to the face. After 28 days of application, the skin properties were evaluated using a Cutometer® to measure the R0 and R5 parameters.

[0139] Effect on skin hardness: After 28 days of application, a decrease in the R0 parameter was observed, which indicates an increase in hardness with Mastocarpus stellatus extract (-7%) compared to placebo. Furthermore, the effects of Mastocarpus stellatus extract were significantly different from the placebo effect, and hardness was found to increase significantly by up to 7-fold during the study, as can be seen from the data below. [Table 9]

[0140] Effect on skin elasticity: After 28 days of application, a significant increase in the R5 parameter was observed with the Mastocarpus stellatus extract compared to D0. With the placebo, a slight increase was observed compared to D0. The measurements further showed that the cream containing the Mastocarpus stellatus extract improved skin elasticity (+ 8%) compared to the placebo cream, as can be seen from the data below. [Table 10]

[0141] Result: Reduction in double chin volume The AEVA HE® was used to measure the effect on double chin volume. After applying an active cream containing 1% Mastocarpus stellatus extract for 28 days, a clear reduction in the volume of the double chin was observed. [Table 11]

[0142] FIG. 3 shows exemplary photographs of AEVA HE® measurements on one volunteer who applied a cream containing 1% Mastocarpus stellatus extract.

[0143] The result: V-shaped improvement In this study, collagen levels were measured with a SiAscope® on the facial oval after applying a cream containing Mastocarpus stellatus extract twice daily for 56 days versus a placebo cream. After 28 and 56 days of application, a significant increase in mean collagen was observed with Mastocarpus stellatus extract of +2.4% and +4.2%, respectively, compared to D0. With placebo, a slight increase was observed after 56 days compared to D0. When comparing products containing Mastocarpus stellatus extract with placebo, it was found that Mastocarpus stellatus extract was able to improve collagen 3.4 times more than placebo after 28 days and 2.8 times more than placebo after 56 days.

[0144] The data is shown in the table below. [Table 12]

[0145] Furthermore, line-field optical coherence tomography (LC-OCT®) was used to describe the dermal fiber network at the level of the facial oval at D0 and D56. LC-OCT® is a non-invasive skin imaging technique that combines high-resolution confocal microscopy to take facial photographs and visualize the effect of collagen.

[0146] After applying a cream containing 1% Mastocarpus stellatus extract for 56 days, it was clearly observed that long collagen fibers appeared in a V-shape on the face. On the other hand, after 56 days of application of the placebo cream, no long collagen tissue was observed. Additionally, elasticity tests were performed in which a 20 g mass suspended by a string and applied in a strip to the facial oval were applied. These tests were performed at D0, D28 and D56. After application of a cream containing 1% Mastocarpus stellatus extract for 28 and 56 days, skin tonus and firmness were found to be increased. For the placebo cream, no effect was observed after 28 and 56 days of application.

[0147] Example 7 Clinical research - skin hydration Prescription In the second set of clinical studies described below, the following cosmetic lotions of INCI formulations were used: Aqua / Water, Mastocarpus stellatus Extract Sodium Benzoate In the placebo lotion, the Mastocarpus stellatus extract was omitted. More specifically, [Table 13]

[0148] Panel Description A single-center study was conducted on 40 volunteers with dry cheek skin and hydration levels <50 a.u. The volunteers were divided into two groups of 20 volunteers each as follows: - Group 1: 20 volunteers with an average age of 47 ± 6 years testing a lotion containing Mastocarpus stellatus extract - Group 2: 20 volunteers with an average age of 48 ± 5 years testing a placebo lotion Volunteers applied a lotion containing 1% Mastocarpus stellatus extract or a placebo lotion every other day for 56 days. Hydration was assessed using Raman spectroscopy.

[0149] result To assess the hydration level of the forearm, the total water content of the stratum corneum was measured in % by Raman spectroscopy after two months of twice-daily application. After applying a lotion containing 1% Mastocarpus stellatus extract for 56 days, it was observed that the total water content of the stratum corneum increased significantly by + 41.9% compared to D0. The placebo showed a significant increase after 56 days compared to D0. In comparison, a lotion containing 1% Mastocarpus stellatus extract improved skin hydration by +10.3% compared to a placebo after 56 days.

[0150] The data can be found in the table below. [Table 14]

[0151] Example 8 Hyaluronidase inhibition test (in tubo) Hyaluronic acid polymers are dissolved by hyaluronidase in the presence of natural substrates. The presence of enzyme inhibitors leads to a decrease in the rate of hydrolysis. The inhibitory effect of the tested extracts on hyaluronidase activity was determined turbidimetrically by measuring the amount of undissolved substrate. The assay was performed in 96-well microplates.

[0152] The following samples were tested: [Table 15]

[0153] 20 μl of each sample was mixed with 10 μl of McIlvaine's buffer (pH 4.6). 20 μl of hyaluronidase and 20 μl of hyaluronic acid were added. Negative controls consisting of vehicle, McIlvaine buffer (pH 4.6), and hyaluronic acid, as well as a positive control containing hyaluronidase, were also tested. In addition, blank samples containing each sample, buffer, and enzyme were prepared and tested with each extraction sample. Samples were tested at various concentrations, with each sample dilution run in triplicate.

[0154] A standard range of disodium cromoglycate (DSCG) was used as the inhibitor standard. The mixture was stirred and left at 37° C. for 40 minutes. Finally, 180 μl of CTAB (cetrimonium bromide) was added to all wells to precipitate unhydrolyzed hyaluronic acid polymers. The mixture was incubated at room temperature for 20 min, and the optical density (OD) at 600 nm was measured using a spectrophotometer.

[0155] The inhibition rate is calculated as follows:

number

[0156] Based on these measurements, IC 50 The concentration of the sample at which hyaluronidase activity was inhibited by 50% was calculated, and the results are shown in the table below: [Table 16]

[0157] As can be seen, the IC50 of the Mastocarpus stellatus extract of the present invention is significantly lower than the IC50 of extracts 1 and 2, revealing the synergistic effect observed with the extract of the present invention.

[0158] Example 9: Anti-glycation activity test (in tube) Glycation is a non-enzymatic chemical reaction that can occur in the core of the dermis. Glucose molecules react with proteins, which leads to the breakdown of the dermis (glycated proteins). The glycated proteins cannot be removed, so they accumulate. Glucose is fixed around collagen and elastin fibers, which stiffen and eventually break (loss of skin elasticity). This process is irreversible. The antiglycation activity test was performed in a 96-well microplate. The same samples as in Example 8 were again tested at various concentrations. Each diluted sample was run in triplicate. A negative control containing sodium phosphate buffer and BSA (bovine serum albumin) and a positive control containing sodium phosphate buffer, BSA and ribose were also tested.

[0159] Finally, two blank samples were prepared and tested with each extraction sample. - Includes phosphate buffer, BSA, and extracts - Contains phosphate buffer, ribose, and extracts A standard range of aminoguanidine was used as an inhibitor benchmark. 40 μl of each sample was mixed with 50 μl of BSA and 10 μl of ribose.

[0160] The mixture was stirred and incubated for 17 h at 37° C. Fluorescence (Fl) was measured at λ excitation = 340 nm and λ emission = 420 nm. The antiglycation activity was calculated as follows:

number

[0161] Based on these measurements, IC 50 The concentration of the sample at which AGE formation was inhibited by 50% was calculated. The results are shown in the table below: [Table 17]

[0162] From the above results, it is clear that a strong synergistic effect was observed for the extract of the present invention.

Claims

1. Method for preparing Mastocaccia stellatus extract, including the following steps: (i) Extract Mastocalpus stellatus with water to obtain a first liquid phase and a first solid phase; (ii) Acid hydrolysis of the first solid phase at high temperature; (iii) Phase separation to obtain a second liquid phase and a second solid phase; and (iv) The first liquid phase and the second liquid phase are combined to obtain a Mastocalpus stellatus extract.

2. The method according to claim 1, wherein the mastocalpus stellatus is dried and / or cut and / or ground before water extraction.

3. The method according to claim 1, wherein the water extraction comprises macerating Mastocalpus stellatus in water, particularly deionized water, at a concentration of about 2 to 10%, more preferably about 4 to 5%, at a temperature of about 10 to 30°C, more preferably about 20°C, for about 5 to 60 minutes, more preferably about 15 minutes.

4. The method according to claim 1, wherein the first liquid phase and the first solid phase are separated by one or more of the following: centrifugation, sieving, and filtration.

5. The method according to claim 1, wherein the acid hydrolysis is carried out at a pH of about 1 to 4, more preferably at a pH of about 1.5 to 2.5, and most preferably at a pH of about 2.

6. The method according to claim 1, wherein the acid hydrolysis is carried out at a temperature of about 50 to 90°C, more preferably at a temperature of about 70 to 90°C, and most preferably at a temperature of about 80°C.

7. The method according to claim 1, further comprising the step of adjusting the pH to about 4 to 8, more preferably about 4.5 to 6, and particularly about 5, after acid hydrolysis.

8. The method according to claim 1, wherein the phase separation of step (iii) comprises one or more of centrifugal separation, filtration, vibrating sieving, and throttling.

9. Mastoc carpus stellatus extract obtained by or available by the method described in any one of claims 1 to 8.

10. A cosmetic composition comprising the Mastocaccia stellatus extract described in claim 9 and a cosmetically acceptable excipient.

11. The cosmetic composition according to claim 10, which is a skincare composition.

12. A method for reducing a double chin, comprising the step of topically applying a Mastocalpus stellatus extract, particularly the Mastocalpus stellatus extract described in claim 9, to the double chin.

13. A method for reducing a double chin, comprising the step of applying the cosmetic composition described in Claim 10 topically to the double chin.

14. Use of Mastocaccia stellatus extract according to claim 9 for the purpose of reducing double chin, promoting lipolysis, increasing skin elasticity, stimulating collagen synthesis, hydrating the skin, reducing the effects of skin aging, and / or preventing the effects of skin aging.

15. Use of the cosmetic composition according to claim 10 for the purpose of reducing a double chin, promoting lipolysis, increasing skin elasticity, stimulating collagen synthesis, hydrating the skin, reducing the effects of skin aging, and / or preventing the effects of skin aging.

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