Cosmetic composition comprising lutein and polysaccharide

Abstract

The present invention relates to an algae-derived composition comprising lutein and a polysaccharide. The invention also relates to the use of said composition as a food, cosmeceutical, cosmetic, nutraceutical, nutraceutical supplement or pharmaceutical composition.

Description

Technical Field

[0001] The present invention relates to the field of natural products and their use in cosmetic methods and in the preparation of food, cosmeceutical, nutritional supplement, nutraceutical or pharmaceutical compositions. Background Art

[0002] Topical administration of vitamin A is commonly used to treat various diseases and for cosmetic purposes, such as use as an anti-aging compound. However, many patients undergoing topical vitamin A therapy experience erythema, scaling, dryness, burning, and itching. These symptoms characterize vitamin A-specific irritant contact dermatitis, often referred to as "vitamin A dermatitis." Clinical manifestations of vitamin A dermatitis vary in intensity, and many patients who would benefit from this type of therapy discontinue treatment due to discomfort. Erythema, scaling / flaking, dryness, burning, and itching characterize irritation.

[0003] These effects appear to be related to activation of retinoic acid receptors by carotenoids, of which there are three retinoic acid receptors (RARs), α, β, and γ, which are members of the nuclear receptor superfamily. It is well established that skin irritation is independent of RARα agonist activity. Since RARγ is more expressed than RARβ, good correlation of stimulation with RARγ has been established in both in vitro and in vivo models.

[0004] There is a need in the art for cosmetic compositions that have similar effects on the skin as Retinol without causing irritation. Summary of the invention

[0005] In a first aspect, the present invention relates to a combination comprising lutein and a polysaccharide, wherein the combination is characterized in that it has

[0006] (i) apocarotenoids in an amount of at least 0.00005% (w / w), and / or

[0007] (ii) Chlorophyll content of less than 0.05%.

[0008] In another aspect, the present invention relates to a method for preparing a combination according to the present invention, comprising the following steps:

[0009] a. preparing a first extract containing lutein and apocarotenoids from a first algal biomass preparation, the preparation being carried out by a process comprising the steps of:

[0010] (i) extracting the first algal biomass with an organic solvent,

[0011] (ii) subjecting the extract obtained in step (i) to saponification conditions,

[0012] (iii) removing negatively charged compounds generated by the saponification performed in step (ii), and

[0013] (iv) treating the fraction obtained in step (iii) under oxidative conditions to convert part of the carotenoids present in said fraction into apocarotenoids,

[0014] b. preparing a second extract containing polysaccharides from a second algal biomass preparation, the preparation being carried out by a process comprising the steps of:

[0015] (i) disrupting the cell walls of the second algal biomass,

[0016] (ii) recovering an alkali-insoluble fraction from the fraction obtained in step (i), and

[0017] (iii) dissolving the alkali-insoluble fraction in an acidic medium, and

[0018] c. Combining the fractions obtained in step a. and step b.

[0019] In a further aspect, the present invention relates to a food, cosmeceutical, cosmetic, nutraceutical, nutritional supplement or pharmaceutical composition comprising the combination according to the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 : UV spectra of some selected lutein derivatives detected by HPLC.

[0021] Figure 2 : shows the dose-response curve for the effect of progesterone on RARα. ATRA is the control agonist for this assay (EC50 2.37 nM). Error bars indicate standard deviation.

[0022] Figure 3 : shows the dose-response curve for the effect of progesterone on RARβ. ATRA is the control agonist for this assay (EC50 0.46 nM). Error bars indicate standard deviation.

[0023] Figure 4 : shows the dose-response curve for the effect of progesterone on RARγ. ATRA is the control agonist for this assay (EC50 0.131 nM). Error bars indicate standard deviation.

[0024] Figure 5: The figure reports the average data of the monitored parameters obtained at each experimental examination. Data are presented as mean ± SEM. Above the error bars, the intra-group (black asterisk) and inter-group (red asterisk) statistical analysis is reported as follows: *p<0.05; **p<0.01; ***p<0.001.

[0025] Figure 6 : The figure reports the average data of the monitored parameters obtained at each experimental examination. Data are presented as mean ± SEM. Above the error bars, the intra-group (black asterisk) and inter-group (red asterisk) statistical analysis is reported as follows: *p<0.05; **p<0.01; ***p<0.001.

[0026] Figure 7 : This graph reports the percentage of subjects related to the effect. If more than 50% of the subjects show improvement, a positive effect of the product on the evaluated parameter is confirmed. DETAILED DESCRIPTION

[0027] The authors of the present invention have found that lutein is a RAR agonist that acts specifically through the RAR-β receptor, with negligible activity on the α and γ receptors. In addition, the authors have found that a composition containing lutein and algae polysaccharides derived from algae is a formulation that can be used when the cosmetic or therapeutic effects of lutein are desired.

[0028] Therefore, in a first aspect, the present invention relates to a combination comprising lutein and a polysaccharide, wherein the combination is characterized in that it has

[0029] (i) apocarotenoids in an amount of at least 0.00005% (w / w), and / or

[0030] (ii) Chlorophyll content of less than 0.05%.

[0031] As used herein, the term combination is used to define any material composition, which includes lutein and polysaccharides (jointly or individually). The term combination includes a combined mixture consisting of a separate formulation of components (i) and (ii), such as a "tank-mix"; and a composition in which all components constitute a part of the same mixture. It should be understood that when the combination is provided as a mixture of components (i) and (ii), the other components (apocarotenoids and chlorophyll, if present) will constitute a part of the same mixture. However, in the case where components (i) and (ii) are provided in a separate container, the remaining components (apocarotenoids and chlorophyll, if present) can constitute a part of a container containing component (i) or a part of a container containing component (ii). In some embodiments, lutein and carotenoids are present in one container, and polysaccharides are present in a second container. Chlorophyll, if present, can be present in the first, second or third container. Whenever the combination is provided as a mixture of all components, it will be understood that, in use, all components will be administered simultaneously. However, in those embodiments in which the different components of the combination are formulated in different containers, the combination can then be formulated for simultaneous, separate or sequential administration thereof. Whenever the components of the combination are provided as a mixture of at least components (i) and (ii), the terms "combination" and "composition" can be used interchangeably.

[0032] The term "lutein" as used herein refers to the compound β,ε-carotene-3,3'-diol or (1R,4R)-4-{(1E,3E,5E,7E,9E,11E,13E,15E,17E)-18-[(4R)-4-hydroxy-2,6,6-trimethylcyclohex-1-en-1-yl]-3,7,12,16-tetramethyloctadeca-1,3,5,7,9,11,13,15,17-nonen-1-yl}-3,5,5-trimethylcyclohex-2-en-1-ol and is defined under CAS number 127-40-2.

[0033] In one embodiment, the composition according to the present invention has a lutein content of at least 0.00005% (w / w). In some embodiments, the lutein content in the composition is between 0.00005% (w / w)-5% (w / w), between 0.05% (w / w)-5% (w / w), or between 2% (w / w)-5% (w / w) - relative to the total weight of the composition.

[0034] The term polysaccharide as used herein refers to a long chain polymer carbohydrate composed of monosaccharide units bound together by glycosidic bonds. Suitable polysaccharides that may be present in the composition according to the present invention include, but are not limited to, starch, cellulose, polysaccharides rich in galacturonic acid and polysaccharides rich in glucosamine.

[0035] As used herein, the term "starch" is used to define a glucose polymer in which pyranose glucose units are linked by α bonds, and it is composed of a mixture of amylose (15-20%) and amylopectin (80-85%). Amylose consists of a straight chain of hundreds of glucose molecules, while amylopectin is a branched molecule consisting of thousands of glucose units (each chain of 24-30 glucose units is an amylopectin unit).

[0036] As used herein, "cellulose" refers to a polymer composed of linear chains of hundreds to thousands of β(1→4) linked D-glucose units.

[0037] As used herein, the term "galacturonic acid-rich polysaccharide" refers to any polysaccharide in which a substantial percentage of the integrated monomers are galacturonic acid. Galacturonic acid is an oxidized form of D-galactose, and its CAS number is 685-73-4. In a preferred embodiment, the galacturonic acid-rich polysaccharide is pectin, which includes:

[0038] - Homogalacturonans, which are composed of linear chains of α-(1-4)-linked D-galacturonic acid.

[0039] -Substituted galacturonans consisting of α-(1-4) linked D-galacturonic acid chains branching from a main chain of D-galacturonic acid residues containing sugar appendages such as D-xylose or D-apiogalacturonan in the case of xylogalacturonan and apiogalacturonan, respectively.

[0040] -Rhamnogalacturonan I (RG-I) pectin, which contains a main chain of repeating disaccharides: 4)-α-D-galacturonic acid-(1,2)-α-L-rhamnose-(1. And contains side chains of various neutral sugars (such as D-galactose, L-arabinose and D-xylose) branching from multiple rhamnose residues.

[0041] - Rhamnogalacturonan II (RG-II), which is a highly branched polysaccharide consisting of a backbone composed exclusively of D-galacturonic acid units.

[0042] The term "glucosamine-rich polysaccharide" refers to any polysaccharide in which a high percentage of the integrated monomers are D-glucosamine. D-glucosamine refers to a glucose molecule in which position 3 is an amine group.

[0043] In some embodiments, the glucosamine-rich polysaccharide is selected from chitin, chitosan, or a combination thereof.

[0044] As used herein, the term "chitin" refers to a β(1-4) polymer of N-acetyl-D-glucosamine, which is a major structural component of invertebrate exoskeletons, insect cuticles, and fungal cell walls. Chitin is a linear, highly crystalline homopolymer of β-1,4N-acetylglucosamine (GlcNAc) composed of β-1,4-linked N-acetylglucosamine residues arranged in antiparallel (α), parallel (β), or mixed (γ, two parallel chains alternating with one antiparallel chain), with the (α) configuration being the most abundant. In most organisms, chitin is cross-linked to other structural components such as proteins and glucans. Chitin is represented by the following formula:

[0045]

[0046] In a specific embodiment, the degree of polymerization of the chitin according to the invention is in the range of 50 to 500, preferably between 100 and 250. In a specific embodiment, the chitin according to the invention exhibits a polydispersity index less than or equal to 2.0, preferably in the range of between 1.0 and 2.0.

[0047] The term "degree of polymerization" as used herein relates to the number of monomer units in a macromolecule or polymer. Methods for determining the degree of polymerization are known to those skilled in the art and are primarily based on the number average degree of polymerization and the weight average degree of polymerization. The number average degree of polymerization is obtained by taking the weighted average of the mole fractions; the weight average degree of polymerization is obtained by taking the weighted average of the weight fractions.

[0048] The term "polydispersity index", also called "dispersity", relates to a measure of the width of the molecular weight distribution. This parameter measures the size heterogeneity of molecules or particles in a mixture. Methods for determining dispersity are known to those skilled in the art and include, but are not limited to, size exclusion chromatography, light scattering measurements, and mass spectrometry (MALDI, electrospray ionization).

[0049] The term also refers to chitin derivatives. Chitin derivatives according to the present invention include but are not limited to chitin phosphate, chitin phosphate sulphate, chitin ethylene glycol, aminoethyl chitin, carboxymethyl chitin, chitosan hydrogel and hydroxyethyl chitin. Chitin derivatives can be obtained from chitin by methods known to those skilled in the art.

[0050] In some embodiments, the polysaccharide is chitin, characterized by a degree of polymerization of 50-500 and / or a polydispersity index less than or equal to 2.0.

[0051] The term "chitosan" as used herein relates to chitin derivatives obtained by deacetylation of solid chitin under alkaline conditions (such as concentrated NaOH) or by enzymatic hydrolysis in the presence of chitin deacetylase. It is a linear polysaccharide consisting of randomly distributed β-(1-4)-linked D-glucosamine (deacetylated units) and N-acetyl-D-glucosamine (acetylated units), characterized by its average molecular weight and its degree of acetylation (the proportion of acetylated glucosamine units along the polymer backbone). Chitosan is represented by the following general formula:

[0052]

[0053] In a specific embodiment, the molecular weight of the chitosan according to the invention is between 10 and 60 kDa, more preferably between 15 and 50 kDa. In a specific embodiment, the degree of acetylation is in the range of 1 to 40%, preferably between 7 and 35%. In a specific embodiment, the degree of polymerization of the chitosan according to the invention is in the range of 50 to 500, preferably between 100 and 250. In a specific embodiment, the chitosan according to the invention exhibits a polydispersity index less than or equal to 2.0, preferably in the range of between 1.0 and 2.0.

[0054] The term "molecular weight" as used herein refers to the average molar mass of a molecule. Unlike small molecules, the molecular weight of a polymer is not a unique value. Instead, a given polymer will have a molecular weight distribution depending, for example, on how the polymer is prepared. Thus, as used herein, the term molecular weight with respect to a polymer refers to the molecular weight or average molecular weight distribution. Methods for determining molecular weight are known to those skilled in the art and include, but are not limited to, 1H-NMR.

[0055] The term "degree of acetylation" as used herein relates to the presence of an acetyl functional group in a compound. The removal of the acetyl functional group is referred to as deacetylation. Methods for determining the degree of acetylation / deacetylation are known to those skilled in the art and include, but are not limited to, nuclear magnetic resonance (NMR) spectroscopy.

[0056] In some embodiments, the polysaccharide is chitosan, characterized by a molecular weight of 10-60 kDa, a degree of acetylation of 1-40%, a degree of polymerization of 50-500 and / or a polydispersity index less than or equal to 2.0.

[0057] Particularly preferred methods for determining the degree of acetylation, average molecular weight and polydispersity of chitosan and chitin according to the present invention are known in the art, for example WO2016096986A1.

[0058] The term "chitosan" also includes chitosan derivatives such as PEG-chitosan (copolymer), chitosan azide, N-phthaloyl chitosan, chitosan-C(6)-MPEG (copolymer), adipic acid chitosan, fumaric acid chitosan, lactic acid chitosan, acetic chitosan, hydrochloric acid chitosan, carboxymethyl chitosan, N-sulfonato-N,O-carboxymethyl chitosan, ascorbic acid chitosan, malic acid chitosan, glutamic acid chitosan, trimethyl chitosan (TMC), aryl chitosan, thiolated chitosan, N-succinyl-chitosan (Suc-Chi), thiosemicarbazone chitosan, N,O-carboxymethyl chitosan (NOCC) and hydroxypropyl chitosan (hydroxyl Chitosan derivatives include propylated chitosan (HPC), N-trimethylene chloride chitosan, phthalic acid chitosan and trimethyl ammonium chitosan. Chitosan derivatives can be obtained from chitosan by methods known to those skilled in the art.

[0059] In some embodiments, the polysaccharide content in the composition is at least 90% (w / w), at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.7%, at least 99.8% or at least 99.9%, all values ​​being expressed in w / w relative to the total weight of the composition.

[0060] In a preferred embodiment, the combination of the present invention comprises lutein and starch. In another preferred embodiment, the combination of the present invention comprises lutein and pectin. In another preferred embodiment, the combination of the present invention comprises lutein and cellulose. In another preferred embodiment, the combination of the present invention comprises lutein and chitin. In another preferred embodiment, the combination of the present invention comprises lutein and chitosan. In another preferred embodiment, the combination of the present invention comprises lutein, chitin and chitosan.

[0061] In some embodiments, the weight ratio of lutein to polysaccharide in the combination according to the present invention is about 0.00001:1; 0.00005:1; 0.0001:1; 0.0005:1; 0.001:1; 0.05:1, 0.1:1 or 1:1.

[0062] The composition of the invention is characterized in that it contains at least 0.00005% (w / w) of apocarotenoids relative to the total weight of the composition.

[0063] As used herein, the term "apocarotenoid" refers to a cleavage product derived from one or more carotenoids. Examples of apocarotenoids include, but are not limited to, abscisic acid, apocarotenal, bisporin, β-ionone, β-cyclocitral, crocin, saffron aldehyde, dihydro-β-ionone, dimethyl-β-cyclocitral, dihydroactinol (DHAD), ethyl ester of β-apo-8'-carotene, α-ionone, pseudoionone, peridinin, apo-10-carotenal (Apo10), apo-12-carotenal (Apo12), apo-14-carotenal (Apo14), apo-14-carotenoic acid (apo-14-carotenoic acid) acid), apo-14-carotene, apo-16-carotenal (vitamin A aldehyde), vitamin A alcohol, retinoic acid (RA), dehydrovitamin A aldehyde, retinoic acid, isotretinoin, alitretinoin, acetyl ester, acetyl ester, adapalene, bexarotene, tazarotene and / or any derivative (including functional derivatives) and / or analogs and / or combinations thereof.

[0064] The content of apocarotenoids in the composition according to the invention can be defined by its percentage content relative to the total weight of the composition or by its percentage content or ratio relative to the lutein present in the composition.

[0065] In one embodiment, the content of apocarotenoids in the composition of the present invention is between 0.00005% and 5% (w / w), more preferably between 0.00005 and 2% (w / w), and even more preferably between 0.0005 and 0.05% (w / w).

[0066] In some embodiments, the weight ratio of lutein to apocarotenoid in the composition of the present invention is about 0.001:1; 0.005:1; 0.01:1, 0.05:1; 0.1:1, 0.5:1, 1:1, 1:0.5; 1:0.1; 1:0.5; 1:0.01; 1:0.05 or 1:0.001.

[0067] The relative content of apocarotenoids with respect to lutein can be determined as follows: separating fractions containing lutein and apocarotenoids by chromatography; determining the areas of the peaks corresponding to lutein and each apocarotenoid; and determining the ratio of the total area of ​​all peaks corresponding to apocarotenoids to the area of ​​the peak corresponding to lutein.

[0068] The term "chlorophyll" as used herein includes porphyrin ring-containing pigments present in cyanobacteria, algae and plants and involved in photosynthesis, including but not limited to chlorophyll A, chlorophyll B and chlorophyll C.

[0069] The composition of the present invention is characterized in that the chlorophyll content it contains is less than 0.05% (w / w) relative to the total weight of the composition. In some embodiments, the chlorophyll content in the composition of the present invention is less than 0.045%, less than 0.04%, less than 0.035%, less than 0.030%, less than 0.025%, less than 0.002%, less than 0.015%, less than 0.01% or less.

[0070] In some embodiments, the combination of the invention is provided such that at least one of components (i) or (ii) is encapsulated in a liposome.

[0071] As used herein, the term "liposome" is used to define various monolayer and multilayer lipid vehicles formed by generating a closed lipid bilayer or aggregate. The liposome may be characterized by having a bilayer membrane-containing vesicle structure (generally comprising phospholipids) and an internal medium (generally comprising an aqueous composition). The liposomes provided herein include monolayer liposomes, multilayer liposomes, and multivesicular (bubble) liposomes. The liposomes provided herein may be positively charged, negatively charged, or neutrally charged. In certain embodiments, the liposome is neutrally charged.

[0072] The liposomes used according to the current embodiment can be prepared by various methods as known to those of ordinary skill in the art.

[0073] The size of liposomes varies according to the synthesis method. The liposomes in the current embodiment can have various sizes. In some embodiments, the liposomes are small, such as an outer diameter of less than about 500nm, less than about 400nm, less than about 100nm, about 90nm, about 80nm, about 70nm, about 60nm or less than about 50nm. Such liposome formulations can also be limited by particle charge (zeta potential) and / or optical density (OD). For example, DOTAP: cholesterol liposome formulations will generally include an OD400 less than 0.45 before nucleic acid is incorporated. Similarly, the total charge of such particles in solution can be limited by a zeta potential of about 50-80mV.

[0074] In certain embodiments, the lipid-based nanoparticles are neutral liposomes (e.g., DOPC liposomes). "Neutral liposomes" or "uncharged liposomes" as used herein are defined as liposomes having one or more lipid components that produce an essentially neutral net charge (essentially uncharged). "Essentially neutral" or "essentially uncharged" means that very few (if any) lipid components in a given population (e.g., a population of liposomes) contain a charge that is not offset by the opposite charge of another component ( / .<., less than 10% of the components contain an unoffset charge, more preferably less than 5%, and most preferably less than 1%). In certain embodiments, neutral liposomes may be primarily comprised of lipids and / or phospholipids that are neutral in themselves under physiological conditions ( / .<., at about pH 7).

[0075] The liposomes of the present invention may comprise phospholipids. In certain embodiments, a single type of phospholipid may be used to create liposomes (e.g., neutral phospholipids such as DOPC may be used to generate neutral liposomes). In other embodiments, more than one phospholipid may be used to create liposomes.

[0076] Phospholipids include, for example, phosphatidylcholine, phosphatidylglycerol, and phosphatidylethanolamine; since phosphatidylethanolamine and phosphatidylcholine are uncharged under physiological conditions (i.e., at about pH 7), these compounds can be particularly useful for generating neutral liposomes. In certain embodiments, the phospholipid DOPC is used to produce uncharged liposomes. In certain embodiments, non-phospholipid lipids (e.g., cholesterol) can be used.

[0077] Phospholipids include glycerophospholipids and certain sphingolipids. Phospholipids include, but are not limited to, dioleoylphosphatidylcholine ("DOPC"), egg phosphatidylcholine ("EPC"), dilauroylphosphatidylcholine ("DLPC"), dimyristoylphosphatidylcholine ("DMPC"), dipalmitoylphosphatidylcholine ("DPPC"), distearoylphosphatidylcholine ("DSPC"), l-myristoyl-2-palmitoylphosphatidylcholine ("MPPC"), l-palmitoyl-2-myristoylphosphatidylcholine ("PMPC"), l-palmitoyl-2-stearoylphosphatidylcholine ("P SPC”), l-stearoyl-2-palmitoylphosphatidylcholine (“SPPC”), dilauroylphosphatidylglycerol (“DLPG”), dimyristoylphosphatidylglycerol (“DMPG”), dipalmitoylphosphatidylglycerol (“DPPG”), distearoylphosphatidylglycerol (“DSPG”), distearoylsphingomyelin (“DSSP”), distearoylphosphatidylethanolamine (“DSPE”), dioleoylphosphatidylglycerol (“DOPG”), dimyristoylphosphatidic acid (“DMPA”), dipalmitoylphosphatidic acid (“DPPG”), PPA”), dimyristoylphosphatidylethanolamine (“DMPE”), dipalmitoylphosphatidylethanolamine (“DPPE”), dimyristoylphosphatidylserine (“DMPS”), dipalmitoylphosphatidylserine (“DPPS”), brain phosphatidylserine (“BPS”), brain sphingomyelin (“BSP”), dipalmitoylsphingomyelin (“DPSP”), dimyristoylphosphatidylcholine (“DMPC”), 1,2-distearoyl-sn-glycero-3-phosphocholine (“DAPC”), 1,2-diperoylphosphatidylcholine (“DPPS”), 1,2-diarachidoyl-sn-glycero-3-phosphocholine ("DBPC"), 1,2-diarachidoyl-sn-glycero-3-phosphocholine ("DEPC"), dioleoylphosphatidylethanolamine ("DOPE"), palmitoyloleoylphosphatidylcholine ("POPC"), palmitoyloleoylphosphatidylethanolamine ("POPE"), lysophosphatidylcholine, lysophosphatidylethanolamine and dilinoleoylphosphatidylcholine.

[0078] In some embodiments, the combination is a composition containing liposomes and polysaccharides, wherein the liposomes encapsulate lutein and apocarotenoids, but do not encapsulate polysaccharides. In this case, the composition of the present invention includes liposomes encapsulating lutein and apocarotenoids, and the polysaccharide is in a soluble form.

[0079] In some embodiments, the combination is a composition comprising liposomes, wherein the liposomes encapsulate lutein, apocarotenoids, polysaccharides, and chlorophyll (if present).

[0080] In some embodiments, the combination is a composition comprising: a first liposome population in which lutein and apocarotenoid are encapsulated; and a second liposome population in which the polysaccharide is encapsulated. If present, the chlorophyll may be present in the first liposome population, in the second liposome population, or in both the first and second liposome populations. In any of these three types of compositions, the chlorophyll may be present in a soluble form in the medium in which the liposomes are suspended.

[0081] In some embodiments, the composition of the present invention may be in the form of a water-soluble formulation or an emulsion.

[0082] Method for preparing the composition of the present invention

[0083] In another aspect, the present invention relates to a method for preparing a combination according to the present invention, comprising the following steps:

[0084] a. preparing a first extract containing lutein and apocarotenoids from a first algal biomass preparation, the preparation being carried out by a process comprising the steps of:

[0085] (i) extracting the first algal biomass with an organic solvent,

[0086] (ii) subjecting the extract obtained in step (i) to saponification conditions,

[0087] (iii) removing negatively charged compounds generated by the saponification performed in step (ii), and

[0088] (iv) treating the fraction obtained in step (iii) under oxidative conditions to convert part of the carotenoids present in the fraction into apocarotenoids,

[0089] b. preparing a second extract containing polysaccharides from a second algal biomass preparation, the preparation being carried out by a process comprising the steps of:

[0090] (i) disrupting the cell walls of the second algal biomass,

[0091] (ii) recovering an alkali-insoluble fraction from the fraction obtained in step (i), and

[0092] (iii) dissolving the alkali-insoluble fraction in an acidic medium, and

[0093] c. Combining the fractions obtained in step a. and step b.

[0094] In a first step, a first algal biomass preparation is processed to obtain the first component of the combination of the invention, namely lutein and apocarotenoids.

[0095] The term "algae" as used herein refers to a large class of various simple, generally autotrophic organisms, ranging from unicellular to multicellular forms, including macroalgae and microalgae, i.e. microscopic algae, generally found in freshwater and marine systems. In the context of the present invention, algae are microalgae, in particular microalgae that produce chitin and / or chitosan.

[0096] As used herein, the term "biomass" includes biological material comprising or derived from living organisms or recently living organisms. By extension, the term includes not only biological material or organisms that constitute organisms, but also biological material or organisms generated in biological processes - spontaneously or non-spontaneously (i.e., stimulated).

[0097] As used herein, the term "algal biomass" refers to a mass of microbial cells.

[0098] In a preferred embodiment, the algal biomass is from a species of the genus Chlorella. Preferably, the microalgae belonging to the genus Chlorella are selected from Chlorella saccharophila, Chlorella vulgaris (CS41), Chlorella sorokiniana or Chlorella sp.

[0099] The first step is divided into four different sub-steps, namely:

[0100] (i) extracting the first algal biomass with an organic solvent,

[0101] (ii) subjecting the extract obtained in step (i) to saponification conditions,

[0102] (iii) removing negatively charged compounds generated by the saponification performed in step (ii), and

[0103] (iv) treating the fraction obtained in step (iii) under oxidative conditions to convert part of the carotenoids present in the fraction into apocarotenoids,

[0104] Step a(i) involves extracting the algal biomass with an organic solvent. In one embodiment, this step is performed without previously treating the biomass to disrupt cell walls or lyse cells.

[0105] As used herein, an organic solvent refers to an organic compound that is different from oil and different from surfactant (e.g., organic solvents are generally non-surface active and non-amphiphilic), is miscible in a liquid extraction medium, and together with the surfactant and water of the liquid extraction medium, can form a liquid extraction medium or a portion of the liquid extraction medium (e.g., a homogenous liquid or liquid phase of the extraction medium) in which the target algae material is dissolved. The term "organic solvent" is readily known to those skilled in the art, but can include chemical solvents such as acetone, acetonitrile, benzene, chloroform, 1,4-dihydroquinone, chlorinated bicarbonate, dichloromethane, dichloro ... In one embodiment, the organic solvent used in the extraction step a(i) is ethanol.

[0106] The general steps of the chemical extraction process may include the steps of mechanically processing a certain amount of algal material, and the steps of chemically extracting the algal material (usually together with a certain amount of endogenous non-target plant material) from the mechanically processed algal material or its derivatives (optionally, in combination with the mechanical processing step). The mechanical processing and chemical extraction processes can be carried out by any useful mechanical processing and chemical extraction techniques, in a useful order. These steps may include contacting the algal material with a liquid extraction medium (or individual components of the liquid extraction medium) as described herein, for example, the liquid extraction medium comprises: a non-aqueous phase - which may contain one or more surfactants, dissolved water and other dissolved exogenous processing components or dissolved components such as acids, bases, aqueous solvents, solubilizers and (although not required or necessarily preferred) a certain amount of dissolved exogenous oil, dissolved exogenous organic solvent or two or more of these. If a sufficiently large amount of water is combined with the non-aqueous phase, the liquid extraction medium can be in the form of a multiphase liquid, which includes a non-aqueous phase and an aqueous phase consisting primarily of water, for example, as multiple phases of an emulsion.

[0107] In some embodiments, an organic solvent can be added to the microbial cell composition, the lysed cell composition, or the demulsified cell composition. In such embodiments, the organic solvent is added at a concentration of less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.1%, or less than 0.05% by volume.

[0108] Step a(ii) comprises subjecting the extract obtained in step (i) to saponification conditions. The term "saponification" as used herein refers to the process of converting fats, oils or lipids into soaps and alcohols by the action of aqueous alkali on the ester bonds present in the fats, oils or lipids.

[0109] In some embodiments, the extract is contacted with the base for a period of time and then heated, agitated, or a combination thereof, until saponification is achieved. In some embodiments, the base has a pKb of 1 to 12, 1 to 10, 1 to 8, 1 to 6, 1 to 5, 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to 5, 3 to 10, 3 to 6, 3 to 5, 4 to 10, 4 to 8, 4 to 6, 5 to 10, or 5 to 8. As used herein, the term "pKt" refers to the negative logarithm of the base association constant, Kb, of the base. Kb refers to the ionization equilibrium constant of the base in water.

[0110] Bases suitable for use in the present invention include, but are not limited to, hydroxide bases (e.g., LiOH, NaOH, KOH, Ca(OH)2, etc., and combinations thereof), carbonate bases (e.g., Na2CO3, K2CO3, MgCO3, etc., and combinations thereof), bicarbonate bases (e.g., LiHCO3, NaHCO3, KHCO3, etc., and combinations thereof), ammonia or quaternary ammonium salts.

[0111] The amount of base used in step (a)(ii) is sufficient to maintain a pH of at least about 7.0 (e.g., at least about 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, or 14.5) in the mixture for the duration of step (a)(ii). In certain embodiments, the reaction is allowed to proceed until substantially (e.g., essentially) all of the chlorophyll in the mixture is converted to an alkali or alkaline earth metal salt of magnesium chlorophyll.

[0112] In some embodiments, the base is added in an amount of about 2% to about 10%, about 2% to about 9%, about 2% to about 8%, about 2% to about 7%, about 2% to about 6%, about 3% to about 6%, about 4% to about 6%, about 5% to about 6%, about 2% to about 5%, about 2% to about 4%, about 2% to about 3%, about 3% to about 5%, about 3% to about 4%, or about 4% to about 5% by weight (or by volume) of the cell culture fluid in order to increase the pH.

[0113] In the method according to the invention, the saponification applied to the extract obtained in step (i) in step (ii) results in the conversion of triglycerides of the membranes derived from the algal biomass into corresponding alcohols and fatty acid salts. Saponification also affects chlorophyll. Chlorophyll is formed by a chlorin ring connected to a phytyl chain via an ester group. When chlorophyll is placed under saponification conditions, the ester bond connecting the chlorin ring to the phytyl is cut, thereby releasing the negatively charged chlorophyll portion. Negatively charged fatty acids and negatively charged chlorin rings can then be removed based on their net charge.

[0114] In numerical terms, the reaction is allowed to proceed until at least about 80%, such as at least about 85%, 90%, 95%, 99%, or 100% complete.

[0115] The progress of this reaction can be monitored by conventional means, such as monitoring the absorbance of the reaction mixture at 405 nm and / or 653 nm, for example at a pH adjusted to between 7.5 and 9.0. It will therefore be appreciated that in certain embodiments, the amount of base and any addition salt used should exceed the amount of chlorophyll present in the extract.

[0116] In one embodiment, saponification is performed by adding an immobilized strong base to the extract. Immobilization is usually achieved by linking the strong base to a solid support.

[0117] Solid supports suitable for the methods disclosed herein can generally have any convenient size and be made of any number of known materials. Preferably, the solid support used in the embodiments disclosed herein can be any suitable type of solid support that provides known binding capacity, resulting in substantially no fluctuation in the amount of binding nucleic acid per fixed amount of solid support. Examples of such materials include: inorganic substances, natural polymers, and synthetic polymers. Specific examples of these materials include: cellulose, cellulose derivatives, acrylic resins, glass, silica gel, gelatin, polystyrene, polyvinyl pyrrolidone, copolymers of vinyl and acrylamide, polystyrene cross-linked with divinylbenzene or the like, polyacrylamide, latex gel, silicon, plastics, nitrocellulose, polystyrene, dextran, rubber, natural sponges, silica gel, controllable microporous glass, metals, cross-linked dextran (e.g., Sephadex TM ), Sepharose TM ) and other solid supports known to those skilled in the art.

[0118] In some embodiments, the solid phase support can include a synthetic polymer support such as polystyrene, polypropylene, substituted polystyrene (e.g., carboxylated or aminated polystyrene), polyamide, polyacrylamide, polyvinyl chloride, etc., or any material that can be used for nucleic acid affinity chromatography. In some embodiments, the solid phase can be a plane, a curved surface, a well, or a part of a microfluidic device.

[0119] In some embodiments disclosed herein, the solid support may include beads. The beads may be any of a variety of shapes, such as spherical, generally spherical, oval, disc-shaped, cubic, amorphous, and other three-dimensional shapes. Various materials, including, for example, resins and polymers, may be used to make beads.

[0120] This allows to remove negatively charged compounds (chlorophyll lacking phytol groups and fatty acids) from the extract by removing fixed alkali from the extract. In one embodiment, strong base is a quaternary amine. As used herein, the term "quaternary amine" refers to a positively charged polyatomic ion comprising a structure NR+4, wherein R is an alkyl or aryl group. Quaternary amine is typically formed by the reaction between the basic nitrogen of the compound and a suitable quaternizing agent (such as, for example, optionally substituted alkyl halide, aryl halide or aralkyl halide, such as methyl iodide or benzyl iodide). Suitable quaternary amines for use in the present invention include but are not limited to salts of tetramethylammonium ions, salts of tetraethylammonium ions, salts of tetrapropylammonium ions or salts of tetrabutylammonium ions. In some embodiments, fixing is achieved by coupling a strong base to a bead.

[0121] According to any of the embodiments described herein, a strong base may be attached to a support.

[0122] In step a(iv), the material obtained in step (iii) is treated under oxidizing conditions. As used herein, the term "oxidizing conditions" refers to contact of a material with an oxidant. The term "oxidant" means an agent (oxidant, oxidizer) or an oxidizing agent (oxide): it is a substance that is capable of oxidizing other substances (in other words, accepting its electrons). Common oxidizing agents are oxygen, hydrogen peroxide, ozone and halogen-containing compounds such as sodium hypochlorite. Oxidizing agents are chemical species that undergo a chemical reaction that obtains one or more electrons. In this sense, it is a component in an oxidation / reduction (redox) reaction. In a second sense, an oxidizing agent is a chemical species that transfers an electronegative atom (usually oxygen) to a substrate.

[0123] The second step (step (b)) of the method according to the present invention is carried out in three different sub-steps, namely:

[0124] (i) disrupting the cell walls of the second algal biomass,

[0125] (ii) recovering an alkali-insoluble fraction from the fraction obtained in step (i), and

[0126] (iii) Dissolving the alkali-insoluble fraction in an acidic medium.

[0127] In the first step, the cells of the second algae biomass are treated to destroy the cell wall, which produces a cracked algae cell composition. As used herein, the terms "crack" ("lyse" and "lysing") refer to the process of breaking the cell wall and / or cell membrane of the cell. The destruction of the cell wall can be carried out by different means, such as mechanical treatment, chemical treatment, enzymatic treatment, physical treatment or a combination thereof. In various embodiments, the cracking occurs by adding an enzyme that can destroy the cell wall of the microbial cell. The cell wall destruction produces a cracked cell composition. The process of the present invention includes cracking the algae cell composition or the algae cell biomass to form a cracked algae cell composition.

[0128] In one embodiment, the disruption of the cell wall is carried out under mild alkaline conditions. This results in the formation of an insoluble fraction containing polysaccharides, and it can be recovered by any means that allows the soluble fraction to be separated from the insoluble fraction, such as filtration or centrifugation. In step (iii) of the process, the alkali-insoluble fraction containing polysaccharides can then be separated by dissolution in an acidic medium, thereby obtaining a polysaccharide-rich fraction, wherein the polysaccharides are present in soluble form.

[0129] The dissolution of alkali-insoluble substances requires a reduction in pH, which can be achieved by using an organic or inorganic acid such as sulfuric acid, nitric acid, phosphoric acid, boric acid, hydrochloric acid, hydrobromic acid, perchloric acid, hypochlorous acid, chlorous acid, fluorosulfuric acid, hexafluorophosphoric acid, acetic acid, citric acid, formic acid or a combination thereof. Due to ease of handling, acids and bases are preferably used in liquid form, particularly in concentrated solutions, wherein the concentration of the acid or base in the solution is preferably in the range of 10 to 55 wt.%, specifically in the range of 20 to 50 wt.%.

[0130] The amount of acid used in step (b)(iii) is sufficient to maintain a pH of at least about 7.0 (e.g., at least about 6.5, 6.0, 5.5, 4.0, 4.5, 3.0, 2.5, 2.0, 1.5, 1.0, 0.5) in the mixture for the duration of step (b)(iii). In certain embodiments, the reaction is allowed to proceed until substantially (e.g., essentially) all of the polysaccharide has been dissolved.

[0131] In some embodiments, the acid is added in an amount of about 2% to about 10%, about 2% to about 9%, about 2% to about 8%, about 2% to about 7%, about 2% to about 6%, about 3% to about 6%, about 4% to about 6%, about 5% to about 6%, about 2% to about 5%, about 2% to about 4%, about 2% to about 3%, about 3% to about 5%, about 3% to about 4%, or about 4% to about 5% by weight (or by volume) of the solution in order to lower the pH.

[0132] Food, cosmeceuticals, cosmetics, nutritional medicine, nutritional supplements or pharmaceutical compositions

[0133] The combination of the present invention may be present in food.

[0134] Food used herein refers to a substance that can be used or prepared for use as food. The term also refers to any substance or product that is intended or reasonably expected to be ingested by humans, whether processed, partially processed or unprocessed. These compositions may also be named "food products", "food compositions", food ingredients or food additives. These compositions may also refer to one or more functions that are beneficial to the body, thereby providing better health and well-being. Therefore, such food products may be intended to prevent and / or treat diseases or pathogenic factors. Therefore, these compositions may also be referred to as functional foods for specific nutritional purposes. Food may be ready-to-eat food. Ready-to-eat food refers to food that does not require dilution with, for example, an aqueous solution suitable for consumption. In principle, the ingredients present in ready-to-eat food are balanced, and it is considered by those skilled in the art that it can be ready-to-eat without adding additional ingredients to the food. Concentrated food refers to such food: one or more ingredients are present in a concentration higher than that present in the ready-to-eat food, so when used, it must be diluted with an aqueous solution suitable for consumption. Non-limiting, illustrative examples of food provided by the present invention include dairy products and their derivatives, for example, fermented milk, yogurt, kephir, curd, cheese, butter, ice cream, dairy-based desserts, etc., and non-dairy products, such as baked products, cakes and pastries, cereals, chocolate, jams, juices, other fruit derivatives, oils and margarines, prepared dishes, etc.

[0135] In another embodiment, the product of the present invention is a nutraceutical product, which comprises the combination of the present invention and a nutraceutical acceptable carrier. In addition, the present invention also relates to the use of the combination of the present invention as an ingredient in a nutraceutical product.

[0136] As used herein, the term "nutraceutical product" refers to a product suitable for human or animal use that contains one or more natural products with therapeutic effects that provide health benefits or have been associated with disease prevention or reduction, and includes dietary supplements that are concentrated natural bioactive products that are normally present (or not present) in food and are presented in a non-food matrix (e.g., capsules, powders, etc.), and that exert a beneficial effect on health greater than that of normal food when taken in doses greater than those present (dosages) in food. Thus, the term "nutraceutical product" includes isolated or purified food products and additives or food supplements that are generally presented in dosage forms for normal oral use, such as capsules, tablets, sachets, drinkable vials, etc.; such products provide physiological benefits or disease protection, generally chronic disease protection. If necessary, the nutritional medical product provided by the present invention may also contain one or more nutritional medical agents (products or substances related to disease prevention or alleviation) in addition to the combination of the present invention, such as flavonoids, ω-3 fatty acids, etc., and / or one or more prebiotics (indigestible food ingredients that stimulate the activity and / or growth of probiotics), such as oligofructose, pectin, inulin, galacto-oligosaccharides, lactulose, human milk oligosaccharides, dietary fiber, etc.

[0137] In another embodiment, the product of the present invention is a cosmeceutical product, which comprises the combination of the present invention and a cosmeceutical acceptable vehicle or carrier. In addition, the present invention also relates to the use of the combination of the present invention as an ingredient in a cosmeceutical product.

[0138] As used herein, the term "cosmeceuticals" refers to products suitable for use on the body or animal body that include one or more cosmeceuticals (functional cosmetics, dermatological products or active cosmetics), i.e., topical hybrid products with cosmetic-pharmaceutical properties, containing active ingredients that produce effects on the user's skin, hair and / or nails at higher and more effective concentrations, thus being at an intermediate level between cosmetics and drugs. Illustrative examples of cosmeceuticals include essential oils, ceramides, enzymes, minerals, peptides, vitamins, etc.

[0139] The present invention relates to a cosmetic composition comprising the combination of the present invention and a cosmetically acceptable carrier or vehicle. The present invention also relates to the use of the combination of the present invention as an ingredient in a cosmetic formulation.

[0140] As used herein, "cosmetic composition" refers to a composition suitable for personal hygiene of humans or animals, or for enhancing natural beauty or changing physical appearance without affecting the structure or function of the human or animal body, including one or more products that provide such effects. If necessary, the cosmetic composition provided by the present invention may also contain one or more cosmetics or cosmetic products in addition to the combination of the present invention, that is, substances or mixtures intended to contact the external parts of the human or animal body (e.g., epidermis, hair system, nails, lips, etc.) or with teeth and buccal mucosa, with the sole or main purpose of cleaning, perfuming, changing its appearance, protecting it, keeping it in good condition or correcting body odor. Illustrative examples of cosmetically acceptable vehicles include products included in the INCI (International Nomenclature of Cosmetic Ingredients) list. The combination of the present invention can be added to various products for cosmetic applications, including cosmetics, creams (e.g., day and night creams, makeup remover creams, foundation creams and sunscreens) for cleaning, protecting, treating or caring for the skin (particularly the face, hands and feet), liquid foundations, makeup remover lotions, protective or skin care body lotions, sunscreen lotions, skin care lotions, gels or foams, such as cleaning, sunscreen and artificial tanning lotions, bath preparations, deodorant compositions, aftershave gels or lotions, depilatory creams and compositions for insect bites and analgesia. The combination of the present invention can take various forms and include, for example, dressings, lotions, solutions, sprays, creams, gels, ointments or similar forms.

[0141] Furthermore, the present invention also relates to a pharmaceutical product comprising the combination of the present invention and a vehicle or carrier suitable for oral, topical or parenteral administration. The present invention also relates to the use of the combination of the present invention as an ingredient in a pharmaceutical composition.

[0142] As used herein, "pharmaceutical composition" refers to physiologically tolerable compositions and molecular entities. Preferably, the term "pharmaceutically acceptable" refers to it has been approved by a state or federal regulatory agency or is included in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, especially in humans.

[0143] Pharmaceutically acceptable carriers or vehicles are well known to those skilled in the art and readily available to the public.

[0144] Depending on the specific mode of administration, the drug product can be formulated as a solid dosage form, a liquid dosage form, an injectable dosage form, or a topical dosage form.

[0145] Solid dosage forms for oral administration can include conventional capsules, sustained-release capsules, conventional tablets, sustained-release tablets, chewable tablets, sublingual tablets, effervescent tablets, pills, suspending agents, powders, granules and gels. Under these solid dosage forms, the active compound can be mixed with at least one inert excipient such as sucrose, lactose or starch. This dosage form can also include other materials (such as in conventional practice) inert diluents, such as lubricants, such as magnesium stearate. In the case of capsules, tablets, effervescent tablets and pills, these dosage forms can also include buffers. Tablets and pills can be made with enteric coatings.

[0146] Liquid dosage forms for oral administration may include emulsions, solutions, suspensions, syrups and elixirs containing pharmaceutically acceptable inert diluents commonly used in the art, such as water. Those compositions may also contain adjuvants, such as wetting agents, emulsifying agents and suspending agents, as well as sweeteners, flavoring agents and fragrances.

[0147] Injectable preparations (e.g., aqueous or oily suspensions, sterile injectables) can be prepared according to known techniques using suitable dispersants, wetting agents and / or suspending agents. Among these acceptable vehicles and solvents, water, Ringer's solution and isotonic sodium chloride solution can be used. Sterile oils are also routinely used as solvents or suspending media.

[0148] For topical administration, the pharmaceutical composition of the present invention may be formulated into creams, gels, hydrogels, lotions, liquids, pomades, spray solutions, dispersions, solid sticks, emulsions, microemulsions and the like, which may be formulated in accordance with conventional methods using suitable excipients such as, for example, emulsifiers, surfactants, thickeners, colorants and combinations of two or more thereof.

[0149] All specific embodiments of the method according to the invention also apply to this aspect of the invention.

[0150] Non-therapeutic use

[0151] As used herein, "antioxidant composition" refers to a composition that reduces the amount of oxidation over a given time compared to oxidation that occurs in the absence of the composition, or refers to a material that increases the time required for a given amount of oxidation to occur compared to oxidation that occurs in the absence of the composition.

[0152] Antioxidant activity can be determined by any known assay such as DPPH (2,2-biphenyl-1-picrylhydrazyl) and ABTS tests. Antioxidant capacity can also be determined by measuring the ability of an antioxidant compound to react with a given free radical, or determining the potential of such a compound to reduce the complex formed between Fe(III) ions and the reagent TPTZ (2,4,6-tripyridyl-s-triazine). Tests that rely on measuring the ability of an antioxidant to react with free radicals include the ORAC test (Oxygen Radical Absorbance Capacity), the TEAC assay (Trolox Equivalent Antioxidant Capacity). In addition, the Radical Scavenging Index (RSI) is a measure of free radical scavenging capacity and can be used to determine antioxidant capacity. In a preferred embodiment, antioxidant capacity is determined by the DPPH method (RSA (Radical Scavenging Activity), free radical scavenging activity).

[0153] All specific embodiments of the method according to the invention also apply to this aspect of the invention.

[0154] Medical Uses

[0155] All specific embodiments of the method according to the invention also apply to this aspect of the invention.

[0156] The present invention is further illustrated by the following examples, which are not to be construed in any way as imposing limitations upon the scope thereof.

[0157] Example

[0158] Material

[0159] Acquisition, purification and encapsulation of Chlorella vulgaris extract

[0160] The dried biomass of Chlorella vulgaris is extracted with alcohol and filtered. The resulting organic extract is purified by a strong basic anionic resin to selectively remove chlorophyll and esterified fatty acids. The resin is separated from the extract by filtration and the purified extract is encapsulated in liposomes by high pressure homogenization after evaporation of the organic solvent.

[0161] Formation of apocarotenoids

[0162] The formation of apocarotenoids was adapted from Henry et al. (J. Agric. Food Chem. 2000, 48, 5008-5013). A gentle stream of synthetic air (20% oxygen) was passed through this encapsulated Chlorella vulgaris fraction until the desired amount of oxidation products was reached - as determined by HPLC. The solution was then acidified before combining with the polysaccharide fraction.

[0163] Extraction of polysaccharide fraction

[0164] The cell wall of Chlorella vulgaris is disrupted under mild alkaline conditions. The supernatant and the alkali-insoluble fraction are separated by centrifugation, and the precipitate is washed several times with water and dried to obtain a polysaccharide fraction. This alkali-insoluble polysaccharide fraction is dissolved in an acidic medium to prepare a composite.

[0165] Preparation of the assembly

[0166] The encapsulated apocarotenoid-containing extract is mixed with a desired amount of the polysaccharide fraction solution to obtain the desired combination.

[0167] Determination of apocarotenoid content

[0168] The encapsulated, oxidized Chlorella fraction sample was extracted with ethyl acetate. The organic solvent was evaporated and the residue was redissolved in acetone. Lutein and chlorophyll contents were quantified by HPLC at 450 nm using their corresponding standard reference compounds. The total apocarotenoid content was calculated using the following formula:

[0169] (Q 包封的黄体素 –Q 氧化的黄体素 ) / Q 包封的黄体素 x 100 = total amount of apocarotenoids %

[0170] Where Q 包封的黄体素 is the lutein content measured by HPLC at 450 nm before oxidation, while Q 氧化的黄体素 It is the content of oxidized progesterone.

[0171] Figure 1 Some examples of UV spectra of apocarotenoids detected at 300-350 nm are shown. These UV spectra are consistent with the spectra described by Kopec et al. (Jl Chromatography B, 1102-1103, 45-51).

[0172] Determination of polysaccharides

[0173] Several methods were used to determine the polysaccharide fraction. Dubois et al. (Anal. Chem. 1956, 28, 3, 350–356) was used as a colorimetric method to achieve sugar determination. For monosaccharide composition, HPLC was used as described in Blanco-Gomis et al. (Anal ChimActa. 2001, 436, 173-180). Glucosamine analysis was performed as described by Yan et al. (Carbohydrate Polymers, 2012, 87, 2, 1774-1778).

[0174] Assays to determine RAR agonist activity

[0175] By Life Technologies The Cell-Based Nuclear Receptor Profiling Service performs an agonist assay for RAR. In this assay, engineered cell lines (UAS-bla HEK 293T) expressing RARα, RARβ, or RARγ are used to test the effects of progesterone. The technology—based on a mammalian-optimized β-lactamase reporter gene (bla) combined with a FRET-enabled substrate—provides a sensitive detection method for the assay.

[0176] Example 1 - Specific agonist activity of progesterone on α, β and γ RARs

[0177] RAR-α-UAS-bla HEK 293T cells were thawed and resuspended in assay medium (DMEM without phenol red, 2% CD-treated FBS, 0.1mM NEAA, 1mM sodium pyruvate, 100U / mL / 100μg / mL Pen / Strep) to a concentration of 312,500 cells / mL. 4μL of 10X serially diluted ATRA (control agonist starting concentration, 10nM) or test compound (lutein) were added to the appropriate wells of 384-well, poly-D-lysine assay plates. 32μL cell suspensions (10,000 cells) were added to each well. 4μL assay medium was added to all wells to reach a final assay volume of 40μL. The plate was incubated for 16-24 hours in a humidified incubator at 37°C / 5% CO2. 8μL of 1μM substrate loading solution was added to each well, and the plate was incubated for 2 hours at room temperature. The plate was read on a fluorescent plate reader. The results are shown in Figure 2 No agonist effect of progesterone could be measured at any concentration below 3.2 μM.

[0178] RAR-βUAS-bla HEK 293T cells were thawed and resuspended in assay medium (DMEM without phenol red, 0.1% BSA, 0.1mM NEAA, 1mM sodium pyruvate, 100U / mL / 100μg / mL Pen / Strep) to a concentration of 312,500 cells / mL. 4μL of 10X serially diluted ATRA (control agonist starting concentration, 10nM) or test compound (lutein) was added to the appropriate wells of a 384-well, TC-treated assay plate. 32μL of cell suspension (10,000 cells) was added to each well. 4μL of assay medium was added to all wells to bring the final assay volume to 40μL. The plate was incubated in a humidified incubator at 37°C / 5% CO2 for 16-24 hours. 8μL of 1μM substrate loading solution was added to each well and the plate was incubated at room temperature for 2 hours. The plate was read on a fluorescence plate reader. The results are shown in Figure 3 A specific RAR-β partial agonist effect of progesterone could be measured at concentrations above 100 nM, reaching approximately 40% activation at 3.16 μM.

[0179] RAR-γ-UAS-bla HEK 293T cells were thawed and resuspended in assay medium (DMEM without phenol red, 2% CD-treated FBS, 0.1 mM NEAA, 1 mM sodium pyruvate, 100 U / mL / 100 μg / mL Pen / Strep) to a concentration of 156,250 cells / mL. 4 μL of 10X serially diluted ATRA (control agonist starting concentration, 10 nM) or test compound (lutein) was added to the appropriate wells of a 384-well, TC-treated assay plate. 32 μL of cell suspension (5,000 cells) was added to each well. 4 μL of assay medium was added to all wells to bring the final assay volume to 40 μL. The plate was incubated in a humidified incubator at 37°C / 5% CO2 for 16-24 hours. 8 μL of 1 μM substrate loading solution was added to each well and the plate was incubated at room temperature for 2 hours. The plate was read on a fluorescent plate reader. The results are shown in Figure 4 No agonist effect of progesterone could be measured at any concentration below 3.2 μM.

[0180] Example 2 - Efficacy of cosmetic products

[0181] The aim of the study was to evaluate the efficacy of an active cosmetic product for the facial area in reducing skin wrinkles and improving skin elasticity, firmness and uneven skin tone compared to a reference product (consisting of 0.3% Retinol). To achieve this objective, a randomized controlled clinical instrumental study was performed on 30 healthy male and female subjects aged over 40 years, showing clinical signs of skin aging such as fine lines / wrinkles in crow's feet and uneven skin tone due to dark spots. Subjects were included to apply the test product on one side of the face and the reference product on the opposite side, according to a previously defined randomization list. Evaluations were performed at baseline (TO) and 7 days (T7), 14 days (T14), 28 days (T28) and 56 days (T56) after product application. Instrumental measurements were performed with the help of non-invasive bioengineering techniques and integrated with clinical analysis performed by a dermatologist and self-assessments filled in by the volunteers.

[0182] Test products : Chlorella extract containing 0.00005% w / w lutein and 0.1% w / w polysaccharide encapsulated in liposomes. Formulated at 1% v / v in Water, Coco-Caprylate / Caprate, Glycerin, Phenoxyethanol, Ethylhexylglycerin, Sodium Acrylate / Sodium Acryloyldimethyl Taurate Copolymer, Hydrogenated Polydecene, Trideceth-6, Sorbitan Laurate, Chlorella Vulgaris Extract, Lecithin, Lactic Acid, Disodium EDTA.

[0183] Reference Products : 0.3% formulated in water, coco-caprylate / caprate, glycerin, phenoxyethanol, ethylhexylglycerin, sodium acrylate / sodium acryloyldimethyl taurate copolymer, hydrogenated polydecene, trideceth-6, sorbitan laurate, caprylic / capric triglyceride, retinol, disodium EDTA.

[0184] How to use : Apply both products twice a day (morning and afternoon) to the allocated half of the face and massage for one to two minutes.

[0185] Figure 5 , Figure 6 and Figure 7 Results of skin profilometry (wrinkle depth), dark spot color intensity assessment, ITA° calculation, and clinical assessment of skin tone evenness are shown.

[0186] in conclusion

[0187] A clinical analysis conducted by dermatologists highlighted:

[0188] At T28, 63% of participants had improvement in dark / age spot color, and at T56, 67% had improvement in dark / age spot color;

[0189] At T28, 53% of the subjects had an improvement in skin tone evenness, and at T56, 60% of the subjects had an improvement in skin tone evenness.

[0190] No significant differences were observed between the two products tested.

[0191] During the study, all subjects tolerated the product well and most of them gave positive evaluations on all aspects investigated (no erythema, edema, scaling, itching or burning sensation was observed, etc.).

Claims

1. A combination comprising lutein and a polysaccharide, wherein the combination is characterized in that it has (i) apocarotenoids in an amount of at least 0.00005% (w / w), and / or (ii) Chlorophyll content of less than 0.05% (w / w).

2. The combination according to claim 1, wherein the polysaccharide is selected from the group consisting of starch, cellulose, polysaccharides rich in galacturonic acid and polysaccharides rich in glucosamine.

3. The combination according to claim 2, wherein the polysaccharide rich in galacturonic acid is pectin or wherein the polysaccharide rich in glucosamine is chitosan, chitin or a combination thereof.

4. The combination according to any one of claims 1 to 3, wherein the content of lutein in the combination is at least 0.00005% (w / w).

5. The combination according to claim 4, wherein the content of lutein is between 0.00005% (w / w) and 5% (w / w).

6. The combination according to claim 5, wherein the content of lutein is between 0.05% (w / w) and 5% (w / w).

7. The combination according to claim 6, wherein the content of lutein is between 2% (w / w) and 5% (w / w).

8. The composition according to any one of claims 1 to 7, wherein the content of polysaccharide in the composition is at least 90% (w / w).

9. The composition according to claim 8, wherein the content of polysaccharide in the composition is at least 96% (w / w).

10. The composition according to claim 9, wherein the content of polysaccharide in the composition is at least 99% (w / w).

11. The combination according to any one of claims 1 to 10, wherein the content of apocarotenoids in the combination is between 0.00005% and 5% (w / w).

12. The combination according to claim 11, wherein the content of apocarotenoid in the combination is between 0.00005% and 2% (w / w).

13. The composition according to claim 12, wherein the content of apocarotenoid in the composition is between 0.0005% and 0.05% (w / w).

14. A combination according to any one of claims 1 to 13, wherein (i) The combination is a composition containing liposomes and polysaccharides, wherein the liposomes encapsulate the lutein and the apocarotenoids, but not encapsulating the polysaccharide, (ii) the combination is a composition comprising liposomes, wherein the liposomes encapsulate the lutein, the apocarotenoid and the polysaccharide, or (iii) The combination is a composition comprising: a first liposome population in which the lutein and the apocarotenoid are encapsulated; and a second liposome population in which the polysaccharide is encapsulated.

15. The composition according to any one of claims 1 to 14, wherein the composition is in the form of a water-based formulation or an emulsion.

16. A method for preparing a combination according to any one of claims 1 to 15, comprising the following steps: a. preparing a first extract containing lutein and apocarotenoids from a first algal biomass preparation, the preparation being carried out by a process comprising the steps of: (i) extracting the first algal biomass with an organic solvent, (ii) subjecting the extract obtained in step (i) to saponification conditions, (iii) removing negatively charged compounds produced by the saponification performed in step (ii), and (iv) treating the fraction obtained in step (iii) under oxidative conditions to convert part of the carotenoids present in said fraction into apocarotenoids, b. preparing a second extract containing polysaccharides from a second algal biomass preparation, the preparation being carried out by a process comprising the steps of: (i) disrupting the cell walls of the second algal biomass, (ii) recovering an alkali-insoluble fraction from the fraction obtained in step (i), and (iii) dissolving the alkali-insoluble fraction in an acidic medium, and c. Combining the fractions obtained in step a. and step b.

17. The method of claim 16, wherein the algal biomass is from a species of the genus Chlorella.

18. The method of claim 17, wherein the Chlorella species is Chlorella vulgaris.

19. The method according to any one of claims 16 to 18, wherein the organic solvent used in extraction step a(i) is ethanol.

20. The method according to any one of claims 16 to 19, wherein the saponification conditions in step a(ii) are created by adding an immobilized strong base to the extract, and wherein the removal of negatively charged compounds in step a(iii) is performed by removing the immobilized base from the extract.

21. The method of claim 20, wherein the strong base is a quaternary amine.

22. A process according to any one of claims 16 to 20, wherein the oxidising conditions used in step a(iv) comprise exposing the material obtained in step a(iil) to oxygen or ozone.

23. The method according to any one of claims 16 to 22, wherein the cell wall disruption is performed under mild alkaline conditions.

24. Combination obtainable by a method according to any one of claims 16 to 23.

25. A food, cosmeceutical, cosmetic, nutraceutical, nutritional supplement or pharmaceutical composition comprising the combination according to any one of claims 1 to 15 or 24 and a vehicle sufficient for formulating the combination into the food, cosmeceutical, nutraceutical, cosmetic, nutritional supplement or pharmaceutical composition.

26. A cosmetic method for preventing and / or reducing the cosmetically adverse effects of aging, the method comprising administering to a subject in need thereof the food, cosmeceutical, nutritional supplement, nutraceutical or cosmetic composition of claim 25.

27. The cosmetic method of claim 26, wherein the cosmetic adverse effects of aging are selected from wrinkles, fine lines, discontinuities, roughness, uneven skin tone, skin sagging, skin spots and / or loss of skin tone brightness.

28. A cosmetic method for improving skin elasticity and firmness, the method comprising administering the food, cosmeceuticals, nutraceuticals, nutritional supplements or cosmetic composition of claim 25 to a subject in need thereof.

29. The cosmetic method of any one of claims 26 to 28, wherein the cosmetic composition is administered topically.

30. Non-therapeutic use of the combination according to any one of claims 1 to 15 or 24 or the food, cosmeceutical, cosmetic, nutritional supplement or nutraceutical composition according to claim 26 as an antioxidant.

31. A pharmaceutical composition comprising the combination according to any one of claims 1 to 15 or 24 and a pharmaceutically acceptable adjuvant.

32. A combination according to any one of claims 1 to 15 or 24 or a pharmaceutical composition according to claim 31 for use in medicine.

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