Method for preparing broadfruit algae extract

Abstract

The invention discloses a method for preparing a broadfruit algae extract and cosmetic application of the extract.

Description

[0001] The present invention relates to a method for preparing a Mastocarpus stellatus extract, the Mastocarpus stellatus extract obtained thereby, a cosmetic composition comprising the Mastocarpus stellatus extract and use of the Mastocarpus stellatus extract in cosmetics.

[0002] Ceratium sutchuenensis, commonly called Ceratium sutchuenensis or false Irish moss, is a species in the Rhodophyceaedivision (red algae). Ceratium sutchuenensis is commonly found on rocks in the mid- and low-tidal zones. It is commonly found on most coasts of Ireland and the United Kingdom. Other recorded locations include: France (English Channel, Northeast Atlantic), Iceland, Faroe Islands, Northern Russia to Rio de Oro, Canada (Newfoundland) to the United States (North Carolina).

[0003] Chondrus crispus grows from a disc-shaped holdfast, and the fronds are grooved, unlike the flat Irish moss (Chondrus crispus). It grows to a height of 10-20 cm and is forked. The leaves are cartilaginous and reddish brown, with a green or purple hue. The mature algae shows reproductive structures developed on upright filaments up to 1 mm in diameter. In color it is reddish brown, purple or bleached.

[0004] According to Michael Guiry, the earliest record of collecting Irish seaweed is attested by the poetry of 12th century monks. In the 2001 Irish Seaweed Aquaculture Market Analysis, the estimated combined national seaweed annual harvest of M. stellatus and Irish moss (C. crispus) was less than 100 tons. M. stellatus is harvested during the gametophyte life stage because the later stages with more sulfated carrageenans are more difficult to remove from its rock. The food and pharmaceutical industries are interested in the antioxidant, anticoagulant and thickening or gelling properties of seaweed. In addition to its health properties and applications, the gelling properties of M. stellatus can produce biodegradable films that can be a sustainable and edible alternative to plastics for food preservation and functional food development.

[0005] EP1743628A1 relates to a cosmetic composition comprising a red algae extract and a carrier, wherein the red algae extract comprises a combination of 25-50 wt% of floridoside and 10-25 wt% of isethionic acid in a mass ratio of 1:5. The composition can be used to hydrate the skin and / or prevent skin aging.

[0006] FR2946878B1 relates to the cosmetic use of floridoglycosides or red algae extracts as melanin production inhibitors.

[0007] Surprisingly, it has now been found that, by using a specific extraction method, it is possible to obtain an extract of Euphorbia latifolia which has lipolytic activity and is able to increase the elasticity of the skin, thus allowing a reduction in heavy chins.

[0008] Therefore, in a first aspect, the present invention provides a method for preparing a Ceratium spp. extract. The method comprises the following steps:

[0009] (i) aqueous extraction of the algae to obtain a first liquid phase and a first solid phase;

[0010] (ii) acidic hydrolysis of the first solid phase at elevated temperature;

[0011] (iii) phase separation to obtain a second liquid phase and a second solid phase; and

[0012] (iv) combining the first liquid phase and the second liquid phase to obtain the Laetiporus lappa extract.

[0013] In a second aspect, the present invention relates to a Dictyophora extract obtained or obtainable by the method of the present invention.

[0014] In a third aspect, the present invention relates to a cosmetic composition comprising the Dictyophora extract of the present invention and a cosmetically acceptable excipient.

[0015] In a fourth aspect, the present invention relates to a method for reducing a heavy chin comprising the step of topically applying a Dictyophora extract, in particular a Dictyophora extract of the present invention or a cosmetic composition of the present invention to said heavy chin.

[0016] In a fifth aspect, the present invention relates to the use of a Laetiporus fulva extract of the present invention or a cosmetic composition of the present invention for reducing a heavy chin, for enhancing lipolysis, for increasing skin elasticity, for stimulating collagen synthesis, for hydrating the skin, for reducing the effects of skin aging and / or for preventing the effects of skin aging.

[0017] The present invention and its preferred features are described in more detail below.

[0018] For the avoidance of doubt, unless the context indicates otherwise, a preference, option, specific feature, etc. indicated for a given aspect, feature or parameter of the present invention should be considered to be disclosed in combination with any and all other preferences, options, specific features, etc. indicated for the same or other aspects, features and parameters of the present invention.

[0019] The method of the present invention comprises aqueous extraction of Dictyophora latifolia to obtain a first liquid phase and a first solid phase.

[0020] The aqueous extraction may be performed on the entire C. latifolia plant. Alternatively, only certain parts of the plant may be used, such as the stalk and / or leaves.

[0021] Prior to the aqueous extraction, the Dictyophora latifolia may be dried and / or processed into smaller pieces, for example by cutting or grinding or crushing or chopping or any other suitable method known to the person skilled in the art.

[0022] Thus, in one embodiment, the algae is dried prior to the aqueous extraction. The algae can be dried by any suitable method, such as in an oven at low temperature (e.g., about 40° C.) or in the sun. Using dried algae facilitates storage and logistics. Alternatively, fresh algae or frozen algae, for example, can also be used.

[0023] In one embodiment, prior to the aqueous extraction, the algae is processed, in particular cut and / or ground into smaller fragments. This improves the efficiency of the extraction process. Preferably, prior to the aqueous extraction, the algae is processed into fragments of about 15 to 20 mm in length (e.g., the fragments may have the following size distribution: 5-10% at >2360 μm, 1000 μm <80-90% <2360 μm, 10-15% at <1000 μm). This allows the extraction process to be optimized while avoiding the extraction of carrageenan in the first extraction step. In the method of the invention, preferably all the carrageenan contained in the algae is subjected to an acidic hydrolysis.

[0024] The aqueous extraction is usually carried out using water, in particular deionized water. Alternatively, regular tap water, or water containing additives such as glycerol, propylene glycol, ethanol or other polar solvents, or buffers, may also be used.

[0025] In one embodiment, the aqueous extraction comprises immersing the algae in water, particularly deionized water.

[0026] The aqueous extraction, in particular the maceration in water, can be carried out with a suitable concentration of Psoralea corylifolia in water.

[0027] In one embodiment, the aqueous extraction comprises soaking the algae in water, particularly deionized water, at a concentration of about 2 to 10%, more preferably about 4 to 5%, such as about 4.8%.

[0028] Throughout the present disclosure, unless otherwise stated, indications of percentage (%) refer to percentage by weight (w / w).

[0029] The aqueous extraction, in particular the maceration in water, can be carried out at room temperature. Alternatively, slightly higher or lower temperatures can also be used.

[0030] In one embodiment, the aqueous extraction comprises macerating the Laevigata at a temperature of about 10 to 30°C, more preferably about 20°C.

[0031] The aqueous extraction, in particular the maceration in water, may be carried out for a suitable period of time depending on the temperature and / or concentration used.

[0032] In one embodiment, the aqueous extraction comprises macerating the Dictyophora latifolia for about 5 to 60 minutes, more preferably about 15 minutes.

[0033] In a specific embodiment, the aqueous extraction comprises soaking the Algae in water, especially in 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 at about 20°C, for about 5 to 60 minutes, more preferably about 15 minutes.

[0034] Any suitable method may be used to separate the first liquid phase and the first solid phase. Preferred methods may vary, for example, depending on the scale of the aqueous extraction, but may also vary depending on other factors such as particle size, viscosity of the liquid phase or available equipment. Centrifugation, sieving and / or filtration are particularly suitable methods.

[0035] In one embodiment, the first liquid phase and the first solid phase are separated by one or more of centrifugation, sieving and filtration.

[0036] After the first phase separation, the first liquid phase can be stored under suitable conditions. For example, during the remaining extraction process, the first liquid phase can be stored at ambient temperature and protected from light for several hours (e.g., about 3-4 h). In addition, the first liquid phase can be stabilized by adding a suitable stabilizer, such as a (polar) solvent. For example, the first liquid phase can be stabilized by adding 20% ​​propylene glycol.

[0037] In particular, acidic hydrolysis of the first solid phase is used to hydrolyze carrageenan contained in the algal material.

[0038] The acidic hydrolysis of the first solid phase can be carried out at a suitable pH. Depending on the desired degree of hydrolysis, a slightly higher or lower pH can be selected.

[0039] In one embodiment, the acidic hydrolysis is performed 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.

[0040] The desired pH can be obtained by adding a suitable acid. Sulfuric acid, hydrochloric acid, citric acid and mixtures thereof are particularly suitable.

[0041] In one embodiment, the acidic hydrolysis comprises adding sulfuric acid, hydrochloric acid, citric acid, or any mixture thereof.

[0042] The ratio of kelp 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.

[0043] The acidic hydrolysis is carried out at elevated temperature. Depending on the desired degree of hydrolysis, higher or lower temperatures can be selected.

[0044] In one embodiment, the acidic hydrolysis is conducted 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.

[0045] The duration of the acidic hydrolysis can also be adjusted according to the desired degree of hydrolysis. The longer the duration, the lower the degree of polymerization.

[0046] In one embodiment, the acidic hydrolysis is carried out for a period of about 1 hour to 3 hours, more preferably about 2 hours.

[0047] For example, the acidic hydrolysis can be stopped by increasing the pH or decreasing the temperature.

[0048] In one embodiment, the process of the present invention further comprises the step of adjusting the pH to a pH of about 4 to 8, more preferably to a pH of about 4.5 to 6, and in particular to a pH of about 5 after said acidic hydrolysis.

[0049] The pH can be adjusted by adding a suitable base, for example an inorganic base such as sodium hydroxide.

[0050] After the acidic hydrolysis and optional pH adjustment, phase separation is performed to obtain a second liquid phase and a second solid phase. The phase separation can be achieved by any suitable means, such as by centrifugation, filtration, vibrating sieving and / or wringing.

[0051] Thus, in one embodiment, the phase separation in step (iii) comprises one or more of centrifugation, filtration, vibratory sieving and wringing.

[0052] Optionally, a suitable stabilizer may be added to the second liquid phase before combining it with the first liquid phase. For example, the second liquid phase may be stabilized by adding a (polar) solvent, such as 20% propylene glycol. Preferably, the same stabilizer is used for both the first liquid phase and the second liquid phase.

[0053] Finally, the first liquid phase and the second liquid phase are combined to obtain the extract of the present invention.

[0054] Optionally, the Dictyophora extract of the present invention may be filtered, for example to at most 0.3 μm, after combining the first liquid phase and the second liquid phase.

[0055] Optionally, after combining the first liquid phase and the second liquid phase, the extract of the present invention may be heated, for example to about 60° C. This may reduce the pink color of the extract by destroying pigments, such as phycoerythrin.

[0056] The following table provides a comparison of the first liquid phase, the second liquid phase and the Dictyophora extract of the present invention:

[0057] First liquid phase Second liquid phase The extract of the present invention Final pH 6.06-6.77 4.84-5.21 5.45-6.10 Dry matter (%) 0.98-1.51 2.19-2.43 1.63-1.78 Floridoglycosides(g / l) 0.87-1.72 0.25-0.74 0.83-0.86 Shinorine (g / l) 0.13-0.20 0.03-0.07 0.07-0.11 Total neutral sugar (g / l) No data No data 10.42

[0058] Depending on the intended use and the desired concentration, the Leptospermum serrata extract may be used as is, or may be diluted or concentrated.

[0059] The L. latifolia extract of the present invention may be stored under suitable conditions, such as at ambient temperature, and preferably protected from light.

[0060] The present invention also relates to a Laetiporus extract obtained or obtainable by the method of the present invention.

[0061] As will be understood by those skilled in the art, the term "obtainable from" as used herein means that the extract can be obtained from a plant or can be isolated from a plant, or can be obtained from an alternative source, such as by chemical synthesis or enzymatic production. Whereas the term "obtained" as used herein means that the extract is directly derived from a plant source.

[0062] The L. latifolia extract of the present invention has been found to have several advantageous cosmetic effects, which have been demonstrated by in vitro and clinical studies (see Examples below).

[0063] In particular, the extract of Psoralea corylifolia was found to reduce heavy chins, enhance lipolysis, increase skin elasticity, stimulate collagen synthesis, hydrate the skin, reduce the effects of skin aging and prevent the effects of skin aging.

[0064] The Leptospermum serrata extract is advantageously applied topically to the skin.

[0065] In a further aspect, the present invention provides a cosmetic composition comprising the extract of the present invention and a cosmetically acceptable excipient. The cosmetic composition of the present invention is intended for topical administration.

[0066] The concentration of the Dictyophora extract in the cosmetic composition should be chosen such that the desired effect is achieved.

[0067] Any excipient commonly used in the preparation of cosmetic preparations for human skin can be used for the present invention. Suitable excipients include, but are not limited to, components that can affect the organoleptic properties, skin penetration and bioavailability of the broad fruit algae extract. More specifically, they include liquids, such as water, oils or surfactants, including those of petroleum, animal, plant or synthetic origin, such as but not limited to peanut oil, soybean oil, mineral oil, sesame oil, castor oil, polysorbate, sorbitan esters, ether sulfates, sulfates, betaines, glycosides, maltosides, fatty alcohols, nonoxynol ethers, poloxamers, polyoxyethylene, polyethylene glycol, dextrose, glycerol, digitonin etc.

[0068] The formulation for topical application to the skin can take any physical form. For example, the cosmetic composition, particularly the skin care composition, can be a liposome composition, mixed liposomes, oleosomes, liposomes, ethosomes, milliparticles, microparticles, nanoparticles and solid-lipid nanoparticles, vesicles, micelles, mixed micelles of surfactants, mixed micelles of surfactants-phospholipids, microspheres, microspheres and nanospheres, lipid spheres, millicapsules, microcapsules and nanocapsules, as well as microemulsions and nanoemulsions, which can be added to achieve greater penetration of the extract of the broad fruit algae.

[0069] Cosmetic compositions, particularly skin care compositions, can be prepared in any solid, liquid or semisolid form that can be applied topically to the skin or by transdermal administration. Therefore, these topical or transdermal preparations include, but are not limited to, creams, multiple 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, microemulsions, emulsions and / or solutions, liquid crystals, anhydrous compositions, aqueous dispersions, oils, emulsions (milks), balms, foams, aqueous or oily lotions, aqueous or oily gels, creams, water-alcohol solutions, water-glycol solutions, hydrogels, liniments, essences, soaps, facial masks, essences, polysaccharide films, ointments, mousses, pomades, pastes, powders, strips, pens and sprays or aerosols (sprays). Includes leave-on and rinse-off preparations.

[0070] Therefore, the present invention also provides skin care compositions.

[0071] The cosmetic composition of the present invention may optionally further comprise other cosmetic active agents, such as anti-aging agents, moisturizers or hydrating agents.

[0072] 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 WO2020 / 201185. The disclosure of WO2020 / 201185 on cosmetic active agents, embodiments thereof and uses thereof is incorporated herein by reference.

[0073] In a further aspect, the present invention also provides a method for reducing a heavy chin, comprising the step of topically applying a Dictyophora extract, in particular a Dictyophora extract according to the present invention or a cosmetic composition according to the present invention, to the heavy chin.

[0074] In a further aspect, the present invention also relates to the use of the extract of the present invention or the cosmetic composition of the present invention for reducing a heavy chin.

[0075] In a further aspect, the present invention also relates to the use of the Euphorbia latifolia extract of the present invention or the cosmetic composition of the present invention for enhancing lipolysis.

[0076] In a further aspect, the present invention also relates to use of the Euphorbia latifolia extract of the present invention or the cosmetic composition of the present invention for increasing skin elasticity.

[0077] In a further aspect, the present invention also relates to the use of the extract of the present invention or the cosmetic composition of the present invention for stimulating collagen synthesis.

[0078] In a further aspect, the present invention also relates to the use of the Euphorbia latifolia extract of the present invention or the cosmetic composition of the present invention for hydrating the skin.

[0079] In a further aspect, the present invention also relates to the use of the Euphorbia latifolia extract of the present invention or the cosmetic composition of the present invention for reducing the effects of skin aging.

[0080] In a further aspect, the present invention also relates to the use of the Leptospermum serrata extract of the present invention or the cosmetic composition of the present invention for preventing the effects of skin aging.

[0081] The present invention is further illustrated by the following non-limiting examples:

[0082] Example 1: Extract of Algae

[0083] A suspension of 4.8% dry matter of A. sphaerocephala in water was prepared (20 g dried algae + 396.67 g osmotic water). The algal material was first cut into 9 mm long pieces with scissors and then soaked for 15 minutes at room temperature (22° C.) under stirring. The biomass (= first solid phase) and the supernatant (= first liquid phase) were separated by filtration (Whatman filter, 40 μm). 1,3-propylene glycol was added to the supernatant at a concentration of 20% of the total amount to obtain extract 1. It was then stored at room temperature in the dark for about 3-4 hours while extract 2 was prepared.

[0084] The previously extracted insoluble part (= first solid phase) was resuspended in permeate water to 4.8% dry matter. The suspension was then stirred, acidified to pH = 2 with 96% sulfuric acid solution and then heated to 80°C. The acid hydrolysis was continued at 80°C for 2 h. After 2 h, 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 solid, and 1,3-propanediol was added to the supernatant (= second liquid phase) at a concentration of 20% of the total amount to obtain extract 2.

[0085] Finally, the two extracts 1 and 2 were combined, stirred and heated to 60° C. Immediately after heating for 30 minutes, the mixture was filtered at 2.5 μm and 0.3 μm in sequence to obtain the Leptospermum serrata extract according to the present invention.

[0086] The extract thus obtained had a pH of about 5.45, a dry mass content of about 1.63%, a Gardner color of about 2.1 and a floridoside content of about 0.86 g / l.

[0087] Example 2: Transcriptomic analysis on human fibroblasts

[0088] Normal human dermal fibroblasts (NHDFs) were seeded in 6-well plates at 300'000 cells per well. After 48 hours of culture in Dulbecco's modified Eagle's medium supplemented with 10% FCS, NHDFs were rinsed twice with phosphate-buffered saline (PBS) and allowed to stand overnight in medium without FCS before stimulation. Cells were stimulated with 0.5% of the extract of the broad algae and compared with untreated conditions. After 6 and 18 hours of stimulation, total RNA was extracted by the Extract-all method (RioDC, Ares M Jr, Hannon GJ, Nilsen TW. Purification of RNA using TRIzol (TRIreagent). Cold Spring Harb Protoc. 2010Jun; 2010(6): pdb.prot5439.doi:10.1101 / pdb.prot5439.PMID:20516177). RNA quality was controlled and reverse transcribed to obtain cDNA. RT-qPCR was performed on specific plates designed to study the transcriptomic expression of different genes involved in skin elasticity of NHDFs with 10 ng of cDNA per well. The results of gene expression obtained with fibroblasts were normalized according to the housekeeping genes PES1 (Pescadillo ribosome biogenesis factor 1), GADD45A (growth arrest and DNA damage inducible alpha) and HMBS (hydroxymethylcholine synthase). Data are expressed as fold change relative to untreated conditions.

[0089] result

[0090] To explore whether the extract of P. latifolia could improve skin elasticity, transcriptomic studies were performed on fibroblasts treated with 0.5% of P. latifolia extract, and the expression of various genes involved in skin elasticity was analyzed.

[0091] After 6 hours of treatment, the extract of the broad algae 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 reduced the expression of HPSE (a gene encoding a protein involved in elastic fiber degradation) by about -53%.

[0092] Interestingly, after 18 h of treatment with the extract of P. latifolia, the expression of FBLN5 and MFAP2 was still significantly increased by +24% and +24%, respectively, and the expression of HPSE was decreased by -40%.

[0093] The extract of D. latifolia also stimulated the expression of genes involved in extracellular matrix structure (eg COL3A1 and CD44) by +33% and +58%, respectively.

[0094] The results are summarized in the following table:

[0095]

[0096] This transcriptomic study validated the extract of P. latifolia as a promising candidate for improving skin elasticity.

[0097] Example 3: Proteomic analysis of human skin explants

[0098] The effects of the extract of P. latifolia on skin elasticity were further evaluated at the proteome level.

[0099] Sample preparation

[0100] Skin explants from young donors (28 years old) and from mature donors (59 years old) remain alive in air-liquid interface. Skin explants from young donors remain untreated, while skin explants from mature donors are topically treated with 1% of the broad fruit algae extract, and compared with untreated conditions. Processing and culture medium (MIL217C from Biopredic International) are updated every day for 5 days. After the stimulation of these 5 days, skin explants are rinsed twice with PBS, and frozen at -80 ℃.

[0101] Sample processing

[0102] The sheared tissue was added to the zirconium oxide bead mixture and 700 μl of iST LYSE buffer (deoxycholate, TCEP and chloroacetamide) was added. The sample was lysed by two rounds of bead beating cycles. The homogenate was transferred to a Diagenode protein extraction tube and the co-extracted nucleic acids and organelles were sheared by microcavitation (Bioruptor Pico, Diagenode). The proteins were dissolved, reduced and alkylated by boiling in iST LYSE buffer (deoxycholate, TCEP and chloroacetamide). The protein concentration was determined by the BCA method (Walker JM. The bicinchoninic acid (BCA) assay for protein quantitation. Methods Mol Biol. 1994; 32: 5-8. doi: 10.1385 / 0-89603-268-X: 5. PMID: 7951748). Peptide extracts were prepared according to the iST method (in Stage Tips). 50 μg of protein were 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 determined using the BCA method.

[0103] LC-MS / MS

[0104] For each sample, 300 ng of peptide were injected in triplicate. Chromatography was performed using an Ultimate 3000 (Di-onex) instrument with a C18 (75 μm × 50 cm, 2 μm material) column, after a 3 min capture step on the pre-column, with a gradient from 2.5% to 35% acetonitrile in 120 min at a flow rate of 300 nL / 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 on the 10 most intense ions of each cycle at a resolution of 17'500 with an accumulation time of 60 ms. 6545 cycles were performed, so an average of 17 cycles per chromatographic peak.

[0105] Protein identification

[0106] Proteins were identified using the SEQUEST-HT algorithm (Tabb DL. The SEQUEST family tree. J Am Soc Mass Spectrom. 2015; 26(11): 1814-1819. doi: 10.1007 / s13361-015-1201-3) against a database of the collected human reference proteome and enzymes used for digestion mined from UNIPROT. Search parameters were: enzyme = trypsin (all); allowed error interval = 2; precursor error tolerance = 10 ppm; fragment error tolerance = 0.02 Da; dynamic modifications = oxidation (M), deamidation (N / Q); protein terminal modifications = acetylation; static modifications = carbamidomethyl (C).

[0107] Using the Percolator algorithm (Spivak M, Weston J, Bottou L, L, Noble WS. Improvements to the percolator algorithm for Peptide identification from shotgun proteomics data sets. J Proteome Res. 2009 Jul; 8(7): 3737-45. doi: 10.1021 / pr801109k. PMID: 19385687; PMCID: PMC2710313), and performed false discovery rate (FDR) determination.

[0108] All spectra reported with confidence less than high by SEQUEST-HT, and therefore considered unidentified, were reprocessed by the MSAmanda 2.0 algorithm against the same database as above. Search parameters were: enzyme = trypsin (all); allowed error interval = 2; precursor error tolerance = 10 ppm; fragment error tolerance = 0.02 Da; dynamic modification = oxidation (M) (P), deamidation (N / Q); protein terminal modification = acetylation, Met-deletion, Met-deletion + acetyl; static modification = carbamidomethyl (C). False discovery rate (FDR) determination was performed using the Percolator algorithm.

[0109] Protein quantification

[0110] Data were processed using Minora and feature mapper for Proteome Discoverer 2.3 software (Thermo Fisher Scientific). Peak integration parameters were: post-acquisition recalibration = true (fine parameter); minimum trace length = 5; minimum number of isotopes = 2; maximum ΔRT of isotopes = 0.2 min; PSM (peptide-spectrum matching) confidence level for integration = high.

[0111] Chromatographic alignment parameters were: RT alignment = true; parameter adjustment = fine; maximum RT offset = 5 min; mass tolerance 10 ppm. Feature mapping parameters were: RT tolerance = auto; mass tolerance = auto; S / N threshold = 2.

[0112] Statistical analyses were performed using the Precursors Ions quantifier node for Proteome Discoverer 2.4 software (Thermo Fisher Scientific).

[0113] General quantification settings are: Peptides to be used = Unique + RAZOR (Unique = peptides not shared by different proteins or proteomes; RAZOR = peptides shared by multiple proteomes, but only used for quantification of proteins with the largest number of unique peptides and the longest amino acid sequence); Proteomes to consider for peptide uniqueness = True; Reject Quan results with missing channels = False. Precursor quantification settings are: Area-based precursor abundance; Minimum number of replicate features = 50% (the peptide must be detected in at least 50% of a set of samples for quantification). Normalization settings are: Total peptide amount (the sum of the abundance values ​​for each injection over all identified peptides is calculated, and the injection with the highest total abundance is used as a reference to correct the abundance values ​​in all other injections by a constant factor for each injection so that the total abundance of all injections is the same at the end).

[0114] Data selection

[0115] A first selection was performed by selecting only proteins that were significantly affected by aging (mature donors vs. young donors) and reversed by the extract of A. luxuriata (treated mature donors vs. untreated mature donors). In this protein selection, proteins involved in the structure and elasticity of the skin dermis were identified: 5 proteins were highlighted.

[0116] result

[0117] First, the effect of skin aging on the proteome was evaluated by comparing young donors (28 years old) with mature donors (59 years old). It was found that the expression of proteins involved in elastic fiber organization such as MFAP4, TIMP1 and FBLN5 (-10%, -60% and -76%, respectively) and other structural proteins of the dermis such as COL1A1 and COL2A1 (-75% each) was significantly reduced. This confirms the negative impact of skin aging on the dermal structure.

[0118] The results compared to younger donors are summarized in the table below:

[0119]

[0120] Secondly, the effect of the extract of C. crassifolia on mature donors whose dermal structure was altered by aging was studied. It was found that the expression of the five proteins mentioned above, which was reduced compared to young donors, increased significantly after 5 days of treatment with the extract of C. crassifolia. In fact, regarding the elastic fiber tissue, the expression of MFAP4, TIMP1 and FBLN5 increased by +64%, +23% and +95%, respectively. In the same way, with the extract of C. crassifolia, the expression of COL1A1 and COL2A1 increased by +17% and +14%, respectively.

[0121] The results compared to mature donors are summarized in the following table:

[0122]

[0123] These results confirm that the extract of P. latifolia has beneficial effects on dermal structure and elastic fiber organization at the protein level.

[0124] Example 4: Analysis of skin biomechanical properties

[0125] Sample preparation

[0126] Skin explants from young donors (19 years old) and from mature donors (49 years old) were kept alive in an air-liquid interface. Skin explants from young donors remained untreated, while skin explants from mature donors were topically treated with 1% of the extract of D. latifolia and compared to untreated conditions. Treatment and culture medium were changed daily for 3 days. After these 3 days of stimulation, skin explants were rinsed twice with PBS and incubated in OCT. TM The samples were cryopreserved in compound mounting medium and cryosectioned at 20 μm thickness.

[0127] Analysis of Young's modulus using atomic force microscopy (AFM)

[0128] The AFM used in this study was a Bioscope Resolve (Bruker) with an added epifluorescence microscope (Leica DMi8). This configuration allows precise positioning of the AFM probe on the sample. This unique combination also allows the acquisition of correlated images from mechanical to fluorescence acquisition.

[0129] QNM (Quantitative Nanomechanical Mapping) was used in this study mode. The AFM probe has a theoretical spring constant of 0.4 N / m and a radius of curvature of <10 nm. Before each use, the probe deflection sensitivity was measured on sapphire, and its spring constant was also calibrated by 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 (PBS1X, batch number: CP20-3404).

[0130] AFM measurements involve acquiring force-volume measurements on the dermis, such as Figure 1 Each pixel of the image corresponds to a force-indentation curve from which the elastic modulus (Ea) is extracted. There may be differences in the number of values ​​from one area to another because some outlier data are suppressed by the software.

[0131] Collagen fiber organization analysis by two-photon microscopy

[0132] A ZEISS LSM880 inverted confocal microscope was used for SHG (second harmonic generation) imaging. The laser used was a coherent two-photon pulsed Cameleon laser. The objective was a "C-Apochromat" water × 40 objective. To image collagen, the sample was excited at a wavelength of 900 nm and the light was collected at 445 nm with a filter. Three large images of 600 μm × 600 μm were prepared for each condition.

[0133] Data analysis was performed using image J and the average gray value on 3 surfaces of 50 × 50 μm size per image was analyzed.

[0134] Collagen fiber orientation was studied using Rozeta software on three areas of 50 x 50 μm per image.

[0135] Result: Skin elasticity restored

[0136] Measurements of the elastic modulus demonstrate the effects of skin aging on both the epidermis and dermis. In fact, the elastic modulus of the 49-year-old donors increased significantly and strongly compared to the 19-year-old donors. This means that the AFM probe requires greater force to deform mature skin compared to young skin, leading to a loss of skin softness with aging.

[0137] However, application of the extract of P. latifolia on mature skin surface significantly reduced the elastic modulus in the epidermis and dermis, demonstrating an improvement in skin softness. The results are summarized in the table below:

[0138]

[0139] Results: Dermal scaffold restoration

[0140] Skin softness / stiffness properties are primarily induced by the dermal scaffold organization. To understand how the elastic modulus is affected by aging, the organization of collagen fibers in the dermis was studied.

[0141] The collagen fibers in young donors were found to present a 2D organization, meaning that the fibers were oriented in at least two directions, thereby constituting a scaffold. On the other hand, the fibers in the dermis from mature donors presented a more isotropic organization, meaning that the fibers were oriented primarily in one direction, thereby showing a loss of dermal scaffolding. This change in collagen fiber orientation with age results in a loss of scaffolding, which may be directly related to the loss of skin suppleness.

[0142] Interestingly, topical application of the extract of P. latifolia on mature donor skin explants resulted in the reorientation of collagen fibers in a 2D organization. This 2D organization favors the reorganization of the dermal scaffold, which could explain the improvement in the biomechanical properties of the skin as revealed by AFM. Figure 2The distribution of collagen fiber orientation in the two dimensions analyzed in the figure is shown. For each direction, the longer the bar, the more fibers are oriented in the corresponding direction.

[0143] Example 5: Lipolytic Activity

[0144] Another factor involved in the formation of a heavy chin is the accumulation of adipose tissue in the chin. To determine whether the extract of D. latifolia could reduce the volume of a heavy chin by reducing adipose tissue content, mature adipocytes were embedded in a 3D matrix.

[0145] 3D Culture of Human Mature Adipocytes

[0146] Mature adipocytes were obtained from subcutaneous adipose tissue from a 30-year-old female with a body weight of 28.7 kg / m 2 of body mass index (BMI) and gave her tissue with informed consent. Mature adipocytes were isolated from subcutaneous adipose tissue after digestion with collagenase. The isolated adipocytes were washed with wash buffer and encapsulated in peptide hydrogels to form 3D adipocyte capsules of 25 μL size. The formation of adipocyte capsules followed an internal standardization protocol to have approximately the same number of adipocytes between capsules. The cells were then incubated at 37°C for 24h for stabilization. Treatment with 0.5% of the wide fruit algae extract was started on day 0, and the culture medium was replaced; and the culture medium was replaced every day until 4 days of culture. At each culture medium change, cell secretions were collected, centrifuged and frozen at -80°C for further analysis. Each culture condition was performed in triplicate.

[0147] Quantification of extracellular secretion of glycerol and adiponectin

[0148] The culture medium was collected after 72 h of treatment. The concentrations of adiponectin and glycerol were assessed by ELISA and colorimetric assay, respectively, using specific kits (adiponectin kit, Duoset, DY1065, R&D Systems; glycerol kit, Randox, GY105) according to the manufacturer's recommendations.

[0149] Result: Glycerol Release

[0150] After 72h of treatment, the evaluation of glycerol released by adipocytes was performed in the adipocyte culture medium. 1 μM isoproterenol (positive control) significantly increased glycerol release by +196% through its lipolytic effect on adipocytes, thus validating the model. 0.5% of the extract of the broad algae also had a significant effect, with a glycerol release of +36% after 72h of treatment, thus demonstrating lipolytic activity.

[0151] The results are summarized in the following table:

[0152]

[0153] Results: Adiponectin secretion

[0154] Adiponectin is another factor secreted by mature adipocytes. It was found that 0.5% of the extract of P. latifolia significantly increased adiponectin secretion by +48% after 72h of treatment, thus confirming its lytic activity on adipose tissue.

[0155] The results are summarized in the following table:

[0156]

[0157] Example 6: Clinical study - skin biomechanical properties, chin reduction and V-shape improvement

[0158] preparation

[0159] For the first set of clinical studies described below, a cosmetic preparation with the following INCI formulation was used:

[0160] AQUA / WATER, CETYL ALCOHOL, GLYCERYL STEARATE, PEG-75 STEARATE, CETETH-20, STEARETH-20, ISODECYL NEOPENTYLATE, ALGAEA LAGONOSA EXTRACT, GLYCERIN, PHENOXYETHANOL, DIMETHICONE, PHENOXYETHANOL, METHYLPARABEN, PROPYLPARABEN, ETHYLPARABEN, Fragrance

[0161] In the placebo composition, the Psoralea corylifolia extract was omitted.

[0162] In more detail:

[0163]

[0164]

[0165] Description of the Panel

[0166] A single-center study was conducted on 44 volunteers with dry skin (skin moisture measurement <70 a.u.) presenting with crow's feet wrinkles and chin ptosis. The volunteers were divided into two groups of 22 volunteers each, as follows:

[0167] - Group 1: 22 women with a mean age of 49 ± 6 years, testing a cream containing an extract of Algae

[0168] - Group 2: 22 women with a mean age of 49 ± 5 years, testing placebo cream

[0169] The study was performed according to the standard operating procedures of the Bio EC and in compliance with the regulations set out in the “Guía para investigaciones con seres humanos” (Guidelines for Research on Human Beings) and the guidelines of the Scientific Committee on Consumer Safety (SCCS).

[0170] The volunteers applied either a cream containing 1% of the Aglaonema serrata extract or a placebo cream in the morning and evening for 28 days.

[0171] During this study, the use Analyze and pass the AEVA as described below The stripe protection is used to analyze the biomechanical properties.

[0172] use Measuring skin biomechanical properties

[0173] The measurement of the mechanical properties of the skin allows the assessment of the functional state of elastic tissue structures (elastic fibers, curvature of the connecting bundles, wrinkles of the stratum corneum) and of tissue structures that behave in a viscous manner (interstitial fluid, internal adhesions).

[0174] Using Courage & Khazaka MPA 580, for research. The measuring principle is based on the suction method: negative pressure is generated in the device and the skin is pulled into the cylindrical hole (2 mm diameter) of the probe. Inside the probe, the penetration depth is determined by an optical measuring system. Each suction phase is followed by a relaxation phase.

[0175] The following procedures were used in this study:

[0176] - Cycle length: 4 seconds (inhale: 2 seconds; relax: 2 seconds)

[0177] - Negative pressure: 450 mbar

[0178] - Chamber diameter: 2mm

[0179] -Measured area: Crow's feet

[0180] At the end of each measurement, the resistance of the skin sucked up by the negative pressure and its ability to return to its original position are displayed as curves. From these curves, the parameters can be calculated:

[0181] - During the suction phase, the skin deformation caused by the negative pressure determines first the elastic resistance and then the viscous component, which together represent the skin firmness.

[0182] - During the relaxation phase, the immediate recovery of the skin determines the complete elasticity of the skin, while the delayed return of the skin to its initial position measures the viscoelastic component.

[0183] The study focused on the following parameters:

[0184] - R0 or Uf, which represents the amplitude of the skin during the suction phase: at equal pressure, the more pliable the skin, the greater the amplitude. This parameter therefore evaluates the viscoelastic dispersion or, in other words, the firmness of the skin.

[0185] - R5 represents the net elasticity (Ur / Ue): the elastic portion of the relaxed area (Ur) divided by the elastic portion of the suction area (Ue).

[0186] These parameters were measured at D0 and D28.

[0187] With AEVA Analysis: Focused chin volume enhancement (V-shaped surgery)

[0188] Based on the patent (US7,821,649), a fringe projection unit combined with stereo imaging technology, The system allows for different measurements ranging from wrinkle reduction to body remodeling. It is designed to quantify the efficacy of cosmetic, aesthetic and dermatological products and treatments.

[0189] In particular, it can be used to assess facial ptosis and chin.

[0190] Statistical analysis

[0191] For in vivo studies, the Shapiro Wilk test was used to verify whether the raw data followed the Gaussian law. In the case of normally distributed data, either unpaired or paired Student's t-test was used to compare the means. In the case of non-normally distributed data, Wilcoxon (paired) and Kruskal-Wallis tests were used for paired and unpaired data, respectively, followed by Mann-Whitney U (unpaired) test.

[0192] Regardless of the statistical test used, the results were considered significant as follows: #p<0.1, *p<0.05, **p<0.01 and ***p<0.001.

[0193] Regarding the analysis of the results of the self-assessment questionnaire and the daily diary, a chi-square test was performed (a dichotomous analysis consisting in comparing the number of relevant answers).

[0194] Results: Increased skin firmness and elasticity

[0195] In the first clinical study, a 1% extract of the phytosporum serrata was applied to the face.TM The skin biomechanical properties of the skin were measured after application of the cream or placebo cream. Skin properties were evaluated, measuring the R0 and R5 parameters.

[0196] Effect on Skin Firmness:

[0197] After 28 days of application, a decrease in the R0 parameter was observed, indicating an increase in firmness with the extract of P. latifolia compared to the placebo (-7%).

[0198] Furthermore, the effects of the extract of the phytospermum officinale were found to be significantly different from the placebo effect, showing a significant increase in firmness of up to 7 times during the study period, as can be seen in the following data:

[0199]

[0200] Effects on skin elasticity:

[0201] After 28 days of application, a significant increase in the R5 parameter was observed with the extract of Psoralea serrata compared to D0. With the placebo, a slight increase was shown relative to D0. The measurements further showed improved skin elasticity (+8%) with the cream containing the extract of Psoralea serrata relative to the placebo cream, as can be seen from the following data:

[0202]

[0203] Result: Reduced chin volume

[0204] AEVA Used to measure the effect on chin volume.

[0205] After 28 days of application of an active cream containing 1% of alginate extract, a significant reduction in the volume of the chin was observed:

[0206]

[0207] Figure 3 Shown is the effect of one of the volunteers who applied a cream containing 1% of the extract of the phytoplankton genus. Illustrative photos of measurements.

[0208] Result: Improvement of V-shape

[0209] During the study, after 56 days of twice-daily application of a cream containing a kelp extract versus a placebo cream, Collagen levels were measured on the facial ellipse.

[0210] After 28 and 56 days of application, a significant increase in mean collagen was observed with the extract of the radiata serrata by +2.4% and +4.2%, respectively, compared to D0. For the placebo, a slight increase was shown after 56 days relative to D0. Comparing the products containing the extract of the radiata serrata to the placebo, it was found that the extract of the radiata serrata was able to improve collagen by 3.4 times after 28 days and by 2.8 times after 56 days.

[0211] The data is shown in the following table:

[0212]

[0213] In addition, line-field optical coherence tomography (LC-OCT) ) to illustrate the dermal fiber network at the level of the facial ellipse at D0 and D56. is a non-invasive skin imaging technique that uses the high resolution of a confocal microscope to take facial photographs in order to visualize the effects of collagen.

[0214] After applying the cream containing 1% of the extract of Psoralea corylifolia for 56 days, the appearance of long collagen fibers on the V-shape of the face was clearly observed.

[0215] On the other hand, no long collagen fibers were observed after 56 days of application of the placebo cream.

[0216] In addition, an elasticity test was performed, which consisted of suspending a 20 g mass by a line and attached to an oval of the face by a strap. This test was performed at D0, D28 and D56.

[0217] It was found that after applying a cream containing 1% of the extract of P. latifolia for 28 and 56 days, the skin had higher tension and tighter firmness.

[0218] For the placebo cream, no effect was observed after 28 and 56 days of application.

[0219] Example 7: Clinical Study - Skin Hydration

[0220] preparation

[0221] For the second set of clinical studies described below, a lotion with the following INCI formula was used:

[0222] Aqua / Water, Algae Extract Sodium Benzoate

[0223] In the placebo lotion, the Psoralea corylifolia extract was omitted.

[0224] In more detail:

[0225] INCI Active ingredients Placebo Water / water 98.50 99.50 Algae Extract 1 / Sodium Benzoate 0.5 0.5

[0226] Description of the Panel

[0227] A single center study was conducted on 40 volunteers with dry skin with hydration levels <50 a.u. on the cheeks. The volunteers were divided into two groups of 20 volunteers each as follows:

[0228] - Group 1: 20 volunteers with an average age of 47 ± 6 years, testing the lotion containing the extract of Algae

[0229] - Group 2: 20 volunteers with an average age of 48 ± 5 years, testing placebo lotion

[0230] Volunteers applied either a lotion containing 1% of the Aglaonema serrata extract or a placebo lotion every other day for 56 days. Hydration was assessed using Raman spectroscopy.

[0231] result

[0232] To assess hydration levels on the forearms, the total water content in % in the stratum corneum was measured by Raman spectroscopy after two months of twice daily application.

[0233] After 56 days of application of the lotion containing 1% of the extract of Psoralea corylifolia, a significant increase of +41.9% in the total water content of the stratum corneum was observed compared to D0. For the placebo, a significant increase was shown after 56 days relative to D0.

[0234] In contrast, after 56 days, the lotion containing 1% Psoralea corylifolia extract showed a +10.3% improvement in skin hydration relative to placebo.

[0235] The data can be found in the following table:

[0236]

[0237] Example 8: Hyaluronidase inhibition test (in tubo)

[0238] In the presence of its natural substrate, the polymer of hyaluronic acid is cleaved by hyaluronidase. The presence of enzyme inhibitors leads to a decrease in the rate of hydrolysis. The inhibitory effect of the test extract on hyaluronidase activity is determined by turbidimetry by measuring the amount of uncleaved substrate.

[0239] The assay was performed in 96-well microplates.

[0240] The following samples were tested:

[0241] sample: illustrate: Extract 1 The first liquid phase obtained in step (i) of the process of the present invention Extract 2 The second liquid phase obtained in step (iii) of the process of the present invention The extract of the present invention In step (iv) of the process of the invention the first and second liquid phases are combined

[0242] 20 μL of each sample was mixed with 10 μL Mcllvaine buffer (pH 4.6). 20 μL hyaluronidase and 20 μL hyaluronic acid were added.

[0243] Negative controls consisting of solvent, Mcllvaine buffer (pH 4.6) and hyaluronic acid were also tested, as well as a positive control that additionally contained hyaluronidase.

[0244] In addition, blank samples containing the corresponding samples, buffer, and enzyme were prepared and tested for each extract sample.

[0245] Samples were tested at various concentrations. Each diluted sample was run in triplicate.

[0246] A standard range of disodium cromolyn (DSCG) was used as an inhibitor reference.

[0247] The mixture was stirred and allowed to stand at 37°C for 40 minutes.

[0248] Finally, 180 μL of CTAB (cetrimonium bromide) was added to all wells to precipitate unhydrolyzed hyaluronic acid polymers.

[0249] The mixture was incubated at room temperature for 20 minutes, and the optical density (OD) at 600 nm was measured using a spectrophotometer.

[0250] The inhibition percentage was calculated as follows:

[0251]

[0252] Based on these measurements, calculate the IC 50 , which is the sample concentration when the hyaluronidase activity is inhibited by 50%. The results are shown in the following table:

[0253] sample: <![CDATA[IC 50 :]]> Extract 1 10.8μl / ml Extract 2 8.4μl / ml The extract of the present invention 4.5μl / ml

[0254] It can be seen that the IC50 of the extract of the present invention is significantly lower than the IC50 of extracts 1 and 2, thereby revealing the synergistic effect observed in the extracts of the present invention.

[0255] Example 9: Anti-glycation activity test (in tubo)

[0256] Glycation is a non-enzymatic chemical reaction that can occur in the heart of the dermis. Glucose molecules react with proteins, which leads to the disintegration of the dermis (glycated proteins). Glycated proteins accumulate because they cannot be eliminated. Glucose is fixed around collagen and elastin fibers, which will harden and eventually break (loss of skin elasticity). This process is irreversible.

[0257] The anti-glycation activity was tested in 96-well microplates.

[0258] The same samples as in Example 8 were tested again at various concentrations. Each diluted sample was run in triplicate.

[0259] 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.

[0260] Finally, two blank samples were prepared and tested for each extract sample:

[0261] -With phosphate buffer, BSA and extract

[0262] -With phosphate buffer, ribose and extract

[0263] A standard range of aminoguanidine was used as an inhibitor reference.

[0264] 40 μL of each sample was mixed with 50 μL BSA and 10 μL ribose.

[0265] The mixture was stirred and incubated at 37°C for 17 h. -激发 =340nm and λ -发射 =420 nm to measure fluorescence (FI).

[0266] The anti-glycation activity was calculated as follows:

[0267]

[0268] Based on these measurements, calculate the IC 50 , that is, the sample concentration at which AGEs formation is inhibited by 50%. The results are shown in the following table:

[0269] sample: <![CDATA[IC 50 :]]> Extract 1 180.4μl / ml Extract 2 177.4μl / ml The extract of the present invention 39.4μl / ml

[0270] The above results reveal a strong synergistic effect observed for the extracts of the present invention.

Claims

1. A method for preparing a Mastocarpus stellatus extract, comprising the following steps: (i) aqueous extraction of the algae to obtain a first liquid phase and a first solid phase; (ii) acidic hydrolysis of the first solid phase at elevated temperature; (iii) phase separation to obtain a second liquid phase and a second solid phase; and (iv) combining the first liquid phase and the second liquid phase to obtain the Laetiporus lappa extract.

2. The method according to claim 1, wherein the Laminaria japonica is dried and / or cut and / or ground prior to the aqueous extraction.

3. The method according to claim 1 or 2, wherein the aqueous extraction comprises soaking the Aglaonema latifolia in water, in particular in 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 at about 20°C, for about 5 to 60 minutes, more preferably about 15 minutes.

4. The method according to any one of claims 1 to 3, wherein the first liquid phase and the first solid phase are separated by one or more of centrifugation, sieving and filtration.

5. The process according to any one of claims 1 to 4, wherein the acidic 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 process according to any one of claims 1 to 5, wherein the acidic 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 any one of claims 1 to 6, further comprising the step of adjusting the pH to a pH of about 4 to 8, more preferably to a pH of about 4.5 to 6, in particular to a pH of about 5 after the acidic hydrolysis.

8. The method according to any one of claims 1 to 7, wherein the phase separation in step (iii) comprises one or more of centrifugation, filtration, vibratory sieving and wringing.

9. An extract of Alpinia scoparia obtained or obtainable by the method according to any one of claims 1 to 8.

10. A cosmetic composition comprising the extract of Caragana serrata according to claim 9 and a cosmetically acceptable excipient. The cosmetic composition according to claim 10 , which is a skin care composition.

12. A method for reducing a heavy chin, comprising the step of topically applying a Dictyophora extract, in particular a Dictyophora extract according to claim 9 or a cosmetic composition according to claim 10 or 11, to the heavy chin.

13. Use of the extract of Euphorbia latifolia according to claim 9 or the cosmetic composition according to claim 10 or 11 for reducing heavy chins, for enhancing lipolysis, for increasing skin elasticity, for stimulating collagen synthesis, for hydrating the skin, for reducing the effects of skin aging and / or for preventing the effects of skin aging.

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