Method for obtaining and stabilising mycosporines-like amino acids and phycoerythrin from biological matrices, composition and cosmetic composition thereof

Abstract

The invention refers to a method for obtaining and stabilising mycosporines-like amino acids and phycoerythrin from biological matrices comprising the following steps: grinding and cell disruption of a biological matrix; extracting the ground biomass with an aqueous eutectic solvent solution; separating a liquid phase comprising mycosporines-like amino acids and phycoerythrin from a solid phase of the extract; removing substantially all the water from the liquid phase and obtaining a concentrated extract. The invention also refers to compositions comprising mycosporines-like amino acids and phycoerythrin obtained have applications in various areas, namely food, pharmaceuticals and cosmetics. The present invention increases the extraction yield and the photo- and thermo-stability of mycosporines-like amino acids and phycoerythrin. The invention provides an extract able to efficiently absorb most of the UV-A, UV-B and HEV blue light radiation.

Description

METHOD FOR OBTAINING AND STABILISING MYCOSPORINES-LIKE AMINO ACIDS AND PHYCOERYTHRIN FROM BIOLOGICAL MATRICES, COMPOSITION AND COSMETIC COMPOSITION THEREOF

[0001] The present invention falls within the technical field of chemical processes for extracting added-value compounds from biological matrices, preferably marine biomass, in particular red macroalgae.

[0002] Mycosporine-like amino acids (MAAs) are found in a variety of organisms, predominantly those exposed to high levels of sunlight, such as marine algae, cyanobacteria, fungi, and some marine invertebrates like corals and sea urchins. Mycosporines-like amino acids are secondary metabolites synthesised by various marine, freshwater and terrestrial organisms, produced as a protective mechanism against UV-A and UV-B radiation. These compounds are low molecular weight molecules (<400 Da), soluble in water, colourless, enriched in nitrogen, and with an absorption maximum in the UV region (310-365 nm), depending on the type of ring they have. Based on the structure of their chromatophore core, mycosporines-like amino acids are classified into two types: aminocyclohexenone ring or aminocyclohexenimine ring, and substituents.

[0003] Mycosporines-like amino acids have already been reported for their high molar coefficient (ε), ranging from 28,100 to 50,000 M-1.cm-1, which results in effective absorption of ultraviolet radiation within specific ranges, which include UV-A radiation between 315 and 400 nm and UV-B radiation between 280 and 315 nm. In cosmetic applications, they thus play a vital role in protecting the skin against damage to deoxyribonucleic acid (DNA) induced directly by ultraviolet radiation or indirectly through the generation of reactive oxygen species, which enhances their application in the cosmetic field. In addition, antioxidant, anti-inflammatory and anti-ageing activities represent other characteristics of cosmetic interest already reported for mycosporines-like amino acids, demonstrating their potential to address issues related to skin ageing and inflammatory conditions.

[0004] Phycobiliproteins are polar photosynthetic proteins found in cyanobacteria, red algae and cyanelles, and are the main photosynthetic pigments in these organisms. They are water-soluble proteins, covalently linked via cysteine amino acids to chromophores called phycobilins, which are open-chain tetrapyrroles organised in supramolecular structures called phycobilisomes. Phycobilisomes, located in the cell stroma, act as light-harvesting complexes. According to their light-absorbing properties and considering that phycobiliproteins differ in their amino acid sequence, the number of chromophores per subunit and the type of chromophore, the phycobiliproteins found in cyanobacteria and red macroalgae are categorised into four groups: phycoerythrin (λmax=490-570 nm); phycocyanin (λmax=610-625 nm); phycoerythrinocyanin (λmax=560-600 nm); allophycocyanin (λmax=650-660 nm). Phycoerythrin is the predominant component and comes in three main forms: B-phycoerythrin (with maximum absorption at 565 and 546 nm and a secondary absorption peak at 499 nm), C-phycoerythrin (with maximum absorption at 565 nm) and R-phycoerythrin (R-FC; with maximum absorption at 565 and 498 nm, and a secondary absorption peak at 540 nm). Among these variants, R-phycoerythrin is the most abundant phycobiliprotein in red algae, generally composed of 6 alpha subunits, 6 beta subunits and 1 gamma subunit.Technical Problems

[0005] The potential of phycoerythrins has already been reported as cancer prevention agents due to their antioxidant and antitumour properties, and their characteristic pink colour, positioning them as a promising alternative to the synthetic pigments widely used in the cosmetics industry. However, it is crucial to consider the potential limitations related to the stability of pigment-protein complexes.

[0006] Given the growing awareness of the origin of ingredients in cosmetic products and consequent adverse allergic reactions to the use of synthetic compounds, there is a need to look for new ingredients that are more environmentally friendly and benign for the skin, where both mycosporines-like amino acids and phycoerythrins can be presented as natural alternatives found in red macroalgae.

[0007] The literature reviews and describes different methodologies for obtaining mycosporines-like amino acids and phycoerythrin from different sources. The preparation of the matrices is the first step and usually involves a mechanical pre-treatment step of the macroalgae, either by maceration or by freeze-drying / grinding with liquid nitrogen, to break the outer cell membrane or cell wall of the algae and thus facilitate the recovery of their intracellular components (A. Ramuet al.). The most common methods used to extract hydrophilic compounds from red macroalgae (in this case, mycosporines-like amino acids and phycoerythrin) mostly include the use of water (M. Martinset al., 2021), buffer solutions (Hemlataet al.) and / or aqueous methanol / ethanol solutions (A. Torreset al.; R. P. Sinhaet al.; K. Whiteheadet al.; N. N. Rosicet al.; Y. Sunet al.). In this context, the manipulation of various parameters, such as temperature, extraction time, pH, solid-liquid ratio and the nature of the solvent, will influence the extraction yield obtained (Y. Sunet al.). However, these processes have disadvantages which are described as: Having low extraction yields; Promoting irreversible denaturation / degradation of compounds resulting in loss of colour (phycoerythrin) and ability to capture UV radiation and High-Energy Visible (HEV) blue light (mycosporines-like amino acids); Having low extraction selectivity (when using aqueous methanol / ethanol solutions); Having problems with low photostability; Having limited commercial use.

[0008] There are also methodologies described that use techniques such as enzyme-assisted extraction (C. Huanget al.), microwave-assisted extraction (C. Juinet al.), pressurised extraction (T.A.V. Carloset al.), ultrasound-assisted extraction (K. Whiteheadet al.; R.D.P. Rodrigueset al.) and extraction with subcritical water (M. Castro-Puyanaet al.). However, although these methods report an increase in yield and / or a reduction in extraction time, they require the use of specific equipment and high operating costs that make the scale-up process more complex, limiting their commercial use. Furthermore, the application of high temperature and pressure in the system, in addition to high energy consumption, can lead to thermal decomposition of the target compounds and consequent loss of their properties (R.P. Rastogiet al.).

[0009] Recently, alternative solvents such as ionic liquids (R.D.P. Rodrigueset al., 2018; R.D.P. Rodrigueset al., 2020; M. Martinset al., 2016) or mixtures of eutectic solvents (Y. Xuet al.) have been highlighted as alternative options for extracting compounds from marine biomass. However, the acceptance of these types of solvents in certain areas is still limited, and their exclusive use does not eliminate the possibility of denaturation / degradation of the compounds when subjected to the temperature variations (Y. L.M. de Souza Mesquitaet al.) required during the industrialization of the process, transport and commercial sale of the products.Solution to Problems

[0010] The present invention solves the problems of the prior art by using an aqueous eutectic solvent solution in the extraction step to increase the extraction yield and the photo- and thermo-stability of mycosporines-like amino acids. Moreover, in the preferred embodiments, after the extraction step, it is carried out a step concerning the removal of the water necessary to guarantee the thermal stability of the phycoerythrin.

[0011] The method for obtaining and stabilising mycosporines-like amino acids and phycoerythrin from biological matrices essentially comprises three main stages: grinding, e.g. milling and cell disruption of a biomass comprising biological matrices; extraction of mycosporines-like amino acids and phycoerythrin with an aqueous eutectic solvent solution; and removal of water from the eutectic solvent. Preferably, the first step comprises the preparation of the biomass with mechanical grinding and subsequent freezing to aid the cell disruption process. Preferably, the second step comprises the solid-liquid extraction of mycosporines-like amino acids and phycoerythrin using an aqueous eutectic solvent solution of neutral pH. Preferably, the water content present in the extract is removed by lyophilisation.Advantageous Effects of Invention

[0012] The present invention overcomes the limitations of the methods already described in the prior art, namely increasing the extraction yield and the photo- and thermo-stability of mycosporines-like amino acids and phycoerythrin using simple, economically viable, and sustainable methodologies that do not require specific equipment or the use of temperature / pressure during the process. Furthermore, the extract comprising mycosporines-like amino acids retains a capacity to absorb UV-A and UV-B electromagnetic radiation, and to absorb HEV blue light.

[0013] The method for obtaining and stabilising mycosporines-like amino acids and phycoerythrin from biological matrices according to the present invention takes place entirely at room temperature and outcomes high extraction yields.

[0014] The mycosporines-like amino acids and phycoerythrin, namely the R-phycoerythrin, obtained have been recognized as natural compounds of great interest for cosmetic applications.

[0015] To promote an understanding of the principles by the embodiments of the present invention, reference will be made to the embodiments illustrated in the figures and to the language used to describe the same. Anyway, it must be understood that there is no intention of limiting the scope of the present invention to the contents of the figures. Any alterations or later changes of the inventive features illustrated herein, and any additional application of the principles and embodiments of the invention shown, which would occur normally for one skilled in the art when reading this description, are considered as being within the scope of the claimed invention.Fig.1

[0016] illustrates a block diagram representing the method for obtaining and stabilising mycosporines-like amino acids and phycoerythrin from biological matrices of the red macroalgaPalmariapalmata;Fig.2

[0017] presents data for (A) extraction efficiency of mycosporines-like amino acids, and (B) extraction efficiency of R-phycoerythrin in %Relative for various aqueous eutectic solvents (25%(w / w)) of water and molar ratio 1:2 of betaine:glycerol, whose starting materials used in their preparation are approved in the cosmetics industry and for phosphate-saline buffer (1 M, pH 7.4), as a conventional process, for both fresh and dried biomasses. The extraction efficiency (%Relative) was calculated relative to the conventional extraction solvent (1 M phosphate-saline buffer, pH 7.4) using fresh biomass, considering an extraction efficiency for this of 100%;Fig.3

[0018] presents data from (A) ultraviolet-visible spectroscopy (UV / VIS), and (B) transmittance of the pure eutectic solvent and the final extract rich in mycosporines-like amino acids and R-phycoerythrin obtained after 35 days of exposure to light and dark conditions at room temperature;Fig.4

[0019] presents data for the viability of HaCat cells, through the MTT assay, after 24 hours of exposure to pure eutectic solvent and the final extract rich in mycosporines-like amino acids and R-phycoerythrin obtained. The results are presented as mean ± standard deviation, calculated considering the viability of untreated cell cultures as 100% (n=3);Fig.5

[0020] presents the chromatographic UHPLC-DAD-ESI / MS spectrum of an aqueous solution done with water from the biomass.

[0021] The present invention refers, in a first aspect, to a method for obtaining and stabilising mycosporines-like amino acids and phycoerythrin from biological matrices characterized by comprising the following steps:

[0022] a) Executing at least one grinding and cell disruption step of at least one biological matrix, obtaining a ground biomass; and

[0023] b) Extracting the ground biomass obtained in Step (a) with an aqueous eutectic solvent solution, obtaining an extract comprising mycosporines-like amino acids and phycoerythrin; and

[0024] c) Separating a liquid phase comprising mycosporines-like amino acids and phycoerythrin from a solid phase comprising the rest of the biomass obtained in Step (b);

[0025] wherein the aqueous eutectic solvent solution comprises water in a range from 5 to 95 % (w / w) in relation to the overall mass of said aqueous eutectic solvent solution; and

[0026] wherein the aqueous eutectic solvent solution comprises at least a first compound and a second compound; and

[0027] wherein the first compound is a hydrogen bond acceptor compound, and the second compound is a hydrogen bond donor compound.

[0028]

[0029] In the preferred embodiments of the invention, the method according to the first aspect comprises the following further step, wherein this further step contributes to improving the thermal stability of the phycoerythrin:

[0030] a) Removing substantially all the water from the liquid phase comprising mycosporines-like amino acids and phycoerythrin, obtained in Step (c), obtaining a concentrated extract comprising mycosporines-like amino acids and phycoerythrin.

[0031]

[0032] Preferably, the aqueous eutectic solvent solution comprises water in a range from 40 to 60 % (w / w), e.g. 50 % (w / w), in relation to the overall mass of said aqueous eutectic solvent solution.

[0033] In the preferred embodiments of the invention, the molar ratio of the first compound and the second compound in the aqueous eutectic solvent solution is in a range from 10:1 to 1:10, even more preferably in a range from 1:1 to 1:5, e.g. 1:2.

[0034] In the preferred embodiments of the invention, the aqueous eutectic solvent solution comprises 10 to 30 % (w / w) of the first compound and 20 to 40 % (w / w) of the second compound.

[0035] In the most preferred embodiments of the invention, the aqueous eutectic solvent solution comprises 10 to 30 % (w / w) of betaine and 20 to 40 % (w / w) of glycerol. As an example of the preferred embodiments of the invention, the aqueous eutectic solvent solution comprises 19.13 % (w / w) of betaine and 30.87 % (w / w) of glycerol.

[0036] The method according to the invention can be applied to the processing of any biological matrix containing mycosporines-like amino acids and phycoerythrin together or individually.

[0037] In the preferred embodiments of the invention, the biological matrix is selected from the group consisting of an algae matrix, a fungi matrix, a bacteria matrix or mixtures thereof. Algae are preferably used, more preferably red macroalgae, and more preferablyPalmariapalmataspecie.

[0038] The feedstock comprising the biomass including biological matrices can be used fresh or dried, preferably fresh to facilitate the grinding step and cell disruption step, which in turn increases the contact area between the aqueous eutectic solvent solution and the ground biomass, resulting in an increase in the extraction yield ().

[0039]

[0040] Biomass preparation – Step (a)

[0041]

[0042] The biomass including biological matrices chosen, whether in dried or fresh form, with a preference for fresh, preferably undergoes immediate processing after harvesting or is stored frozen at a temperature in the range of -20ºC to -80ºC. In the most preferred embodiments, the biomass including biological matrices is stored frozen after being washed with running distilled water. It is then used in Step (a) after the thawing process.

[0043] In the preferred embodiments of the invention, a first grinding and cell disruption step is executed in Step (a) by grinding the biological matrix with liquid nitrogen, wherein a mechanical second grinding step is executed after said first grinding and cell disruption steps. More preferably, a fresh biological matrix is submitted to Step (a) to increase the contact area between the aqueous eutectic solvent solution and the ground biomass.

[0044] The mechanical grinding step can be executed in a bead mill, a wet ball mill, a rotor-stator mill, a hammer mill, a cryogenic mill or a disc mill.

[0045] In the preferred embodiments of the invention, after Step (a), the ground biomass is stored frozen at a temperature in the range of -20ºC to -80ºC.

[0046] Preferably, the first grinding and cell disruption with liquid nitrogen is executed in a temperature in the range of 4ºC to 40ºC, more preferably in a temperature in the range of 10ºC to 30ºC.

[0047]

[0048] Solid-liquid extraction ofmycosporines-like amino acids and phycoerythrin – Step (b)

[0049]

[0050] Step (b) is carried out from the ground biomass resulting from Step (a), where contact is promoted between the ground biomass and an aqueous eutectic solvent solution, preferably of neutral pH.

[0051] Preferably, the pH of the aqueous eutectic solvent solution is in the range from 5 to 8, more preferably in the range from 6.5 to 7.5, e.g. 7.0.

[0052] Aqueous solutions of various eutectic solvents to be used in the solid-liquid extraction step have been tested with a view to their approval in the cosmetics industry (). These preferably have a pH in the range from 6.5 to 7.5, e.g. 7.0.

[0053] In the preferred embodiments of the invention, the first compound, which is comprised in the aqueous eutectic solvent solution, is selected from the group consisting of 1,2-propanediol, citric acid, decanoic acid, lauric acid, lactic acid, capric acid, malic acid, myristic acid, octanoic acid, oleic acid, palmitic acid, nonanoic acid, caffeic acid, 2,3-dihydroxysuccinic acid, alanine, arginine, betaine, fructose, L-carnitine, L-glucose, L-glutamic, nicotinamide, proline, trimethylglycine, glycerol, d-glucose, maltose, sucrose, xylose, thymol, menthol, glycine, histidine, lysine, ornithine, serine, threonine, potassium carbonate, sodium propionate, sodium acetate, choline chloride, choline acetate, acetyl tributyl citrate, triethyl citrate, tributyl citrate, L-cystine, ethylacetate, glycerol, glycerol triacetate, glycerol monostearate, ethyl oleate or mixtures thereof.

[0054] In the preferred embodiments of the invention, the second compound, which is comprised in the aqueous eutectic solvent solution is selected from the group consisting of glycerol, betaine, ethanediol, 1,2-propanediol, 1,3-propanediol, 1,6-hexanediol, panthenol, trehalose, hexadecanoic acid, lactic acid, lauric acid, octanoic acid, oleic acid, palmitic acid, stearic acid, succinic acid, decanoic acid, nonanoic acid, pyruvic acid, acetic acid, ascorbic acid, aspartic acid, benzoic acid, citric acid, fumaric acid, leucine acid, malic acid, propionic acid, tartaric acid, caffeic acid, L-glucose, sorbitol, xylitol, mannitol, D-glucose, fructose, sucrose, alanine, arginine, cysteine, glutamic acid, glutamine, glycine, histidine, inositol, lysine, nicotinic acid, serine, proline, 2-methoxyphenol, quercetin, vanillic acid, vanillin, guaiacol, gallic acid, thereonine, menthol, thymol, leucine or mixtures thereof.

[0055] In the preferred embodiments of the invention, the ratio between both the first compound and the second compound in the aqueous eutectic solvent solution is in the range of 10:1 to 1:10, more preferably in the range of 1:1 to 1:5.

[0056] Preferably, the extraction in Step (b) is executed by mixing the biomass obtained in Step (a) with the aqueous eutectic solvent solution in a proportion in the range of 0.0001 to 0.50 grams of biomass per mL of aqueous eutectic solvent solution.

[0057] Preferably, the extraction in Step (b) is executed in a temperature in the range of 4ºC to 50ºC, even more preferably in the range from 10ºC to 35ºC.

[0058] In the most preferred embodiments, the extraction is executed in a stirred mixing system operating at a range of 5 to 300 rpm, even more preferably in the range of 50 to 80 rpm, e.g. 80 rpm.

[0059] In the most preferred embodiments, the extraction time is in the range of 1 min to 24 hours, more preferably in the range of 1 min to 6 hours, even more preferably in the range of 1 min to 1 hour, even more preferably in the range of 5 min to 10 minutes.

[0060]

[0061] Separating a liquid phase comprisingmycosporines-like amino acids and phycoerythrin from a solid phase of the diluted extract – Step (c)

[0062]

[0063] After the solid-liquid extraction period, all the content not soluble in the aqueous eutectic solvent solution is separated, either by filtration or centrifugation. The liquid fraction, which comprises mycosporines-like amino acids and R-phycoerythrin, is processed in Step (d).

[0064] Preferably, the solid-liquid separation in Step (c) is at least one of the group consisting of a filtration or a centrifugation.

[0065]

[0066] Removing substantially all the water from the liquid phase comprisingmycosporines-like amino acids and phycoerythrin – Step (d)

[0067]

[0068] The liquid fraction obtained from Step (c) is subjected to a step of water removal to eliminate substantially all the water content. As a result of this step, a viscous liquid comprising mycosporines-like amino acids and phycoerythrin is obtained.

[0069] In the most preferred embodiments, a lyophilisation is executed in Step (d) for removing substantially all the water from the liquid phase comprising mycosporines-like amino acids and phycoerythrin.

[0070] Preferably, the mycosporines-like amino acid is at least one of the group consisting of shinorine, porphyra-334, palythine, palythinol, asterina-330, mycosporine-glycine, mycosporine-taurine, usujirene, mycosporine-glutaminol-glucoside, mycosporine-glutaminol, mycosporine-glutamic acid-glycine, mycosporine-glycine, mycosporine-methylamine-threonine, mycosporine-methylamine-serine, mycosporine-serinol, porphyra-331 or gadusol.

[0071] Preferably, the phycoerythrin is R-phycoerythrin.

[0072] In the preferred embodiments according to the method for obtaining and stabilising mycosporines-like amino acids and phycoerythrin from biological matrices, the composition comprising mycosporines-like amino acids and phycoerythrin, obtained in Step (b) has a concentration of mycosporines-like amino acids in the range from 5 mg to 200 mg of mycosporines-like amino acids per g of biomass, even more preferably in the range from 20 mg to 40 mg of mycosporines-like amino acids per g of biomass. Furthermore, the composition comprising mycosporines-like amino acids and phycoerythrin, obtained in Step (b) has a concentration of phycoerythrin in the range from 0.05 mg to 10 mg of phycoerythrin per g of biomass, even more preferably in the range from 0.2 mg to 1 mg of phycoerythrin per g of biomass.

[0073] In other preferred embodiments, the composition comprising mycosporines-like amino acids and phycoerythrin, obtained in Step (d), after removing substantially all the water from the liquid phase comprising mycosporines-like amino acids and phycoerythrinhas, has a concentration of mycosporines-like amino acids in the range from 10 mg to 400 mg of mycosporines-like amino acids per g of biomass, even more preferably in the range from 40 mg to 80 mg of mycosporines-like amino acids per g of biomass. Furthermore, the composition comprising mycosporines-like amino acids and phycoerythrin, obtained in Step (d) has a concentration of phycoerythrin in the range from 0.1 mg to 20 mg of phycoerythrin per g of biomass, even more preferably in the range from 0.4 mg to 2 mg of phycoerythrin per g of biomass.

[0074] The present invention refers, in a second aspect, to a composition comprising mycosporines-like amino acids and phycoerythrin, prepared by the method defined in the first aspect.

[0075] The present invention refers, in a third aspect, to a cosmetic composition, which comprises thecomposition comprising mycosporines-like amino acids and phycoerythrin, as defined in the second aspect.

[0076] The method for obtaining and stabilising mycosporines-like amino acids and phycoerythrin, according to the present invention provides a highly efficient process in terms of increasing the extraction yield by 43.1% for mycosporines-like amino acids and 28.0% for R-phycoerythrin (), while at the same time guaranteeing an increase in the photo- and thermo-stability of mycosporines-like amino acids and R-phycoerythrin compared to conventional processes, i.e. phosphate-salt buffer solution (pH 7.4). Of particular note is the increase in t(1 / 2)of mycosporines-like amino acids from 17.42 hours with conventional methods to 98.33 hours (4.1 days) at 75ºC in the dark, with a maximum of 855 hours (35.6 days) reached in the situation of no exposure to light and a temperature in the range of 20ºC to 25ºC. The results ofrefer to an aqueous eutectic solvent solution including the overall amount of the first compound and the second compound with a concentration of 25%(w / w) of water in relation to the overall mass of said aqueous eutectic solvent solution and a molar ratio 1:2 of betaine:glycerol, which in mass corresponds to 19.13 % (w / w) of betaine and 30.87 % (w / w) of glycerol.

[0077] A further advantage of the method according to the present invention refers to the feasibility of replacing synthetic ultraviolet radiation filters with mycosporines-like amino acids and replacing colour pigments by R-phycoerythrin in cosmetic industry products.

[0078] The method according to the invention also boosts sustainability by recovering compounds from renewable biomass and by using eutectic solvents with desirable properties in the formulation of cosmetic products, thus avoiding the step of separating solvents from the compounds extracted, using the solvents as part of the ingredients of the formulation, replacing and / or decreasing the amount of certain ingredients with properties similar to the eutectic solvents used.

[0079] describes a preferred embodiment of the method for obtaining and stabilising mycosporines-like amino acids and phycoerythrin, according to the present invention, which comprises the following steps:

[0080] a) a first grinding and cell disruption step of Red macroalga with liquid nitrogen, and a second mechanical grinding step, e.g. using a coffee grinder, wherein the ground biomass is washed with water, e.g. distilled water;

[0081] b) Extracting the ground biomass with an aqueous eutectic solvent solution comprising betaine and glycerol, obtaining an extract comprising mycosporines-like amino acids and phycoerythrin;

[0082] c) Separating the liquid phase comprising mycosporines-like amino acids and phycoerythrin from the solid phase of the extract by a centrifugation step;

[0083] d) Removal of the water present in the liquid phase by lyophilisation.

[0084]

[0085] After detailed analysis of the sample of mycosporines-like amino acids and R-phycoerythrin obtained from Step (d), and comparison with a sample obtained from a conventional methodology using a conventional solvent, phosphate-buffered saline (pH 7.4) as the extraction solvent, it was proven:

[0086] i) the presence of mycosporines-like amino acids and phycoerythrin (Figure 3A) with concentrations of 59.8 mg of mycosporines-like amino acids per g of biomass and 1.1 mg of phycoerythrin per g of biomass after lyophilization, respectively;

[0087] ii) the predominant presence of 53.96 % of Porphyra-334 and 30.34 % of mycosporine-methylamine-serine (Table 1 and) in the extract obtained using water as solvent;

[0088] iii) higher extraction yield shown in %Relative ();

[0089] iv) increased photo- and thermo-stability of mycosporines-like amino acids and R-phycoerythrin compared to the product obtained using the conventional process, i.e. using only phosphate-buffered saline (pH 7.4) as the extraction solvent (Tables 1 and 2);

[0090] v) preservation of the ability to absorb most of the incident ultraviolet radiation, UV-A and UV-B (Figure 3B) and HEV blue light radiation (Figure 3B);

[0091] vi) moderate cytotoxicity of both the eutectic solvent and the extract rich in mycosporines-like amino acids and R-phycoerythrin ().

[0092] Examples

[0093] Example 1. Biomass preparation

[0094] Step (a) of the method according to the invention was carried out as described.

[0095] 100 g ofPalmariapalmatawere carefully inspected to remove residues of other algae species and washed with running water followed by distilled water. The excess of water was removed, and the biomass was ground with liquid nitrogen. Finally, it was ground with a coffee grinder for around 10 seconds and then frozen at -20ºC.

[0096] Example 2. Solid-liquid extraction ofmycosporines-like amino acids and R-phycoerythrin

[0097] The biomass used in Example 1 was thawed and used following the solid-liquid extraction.

[0098] 1 mL of a solution containing 50% (w / w) of water and betaine:glycerol in a molar ratio of 1:2 was added to 0.008 g of biomass, with constant stirring at 80 rpm, at 20-25ºC for 5 minutes.

[0099] The biomass was then separated from the aqueous solution of eutectic solvent (betaine:glycerol) using conventional separation methods, namely centrifugation at 12,000 g at 20-25ºC for 15 minutes.

[0100] The combination of the eutectic solvent betaine:glycerol was chosen on the basis of its higher extraction yield compared to the other eutectic solvents tested and the so-called conventional processes, like the one using a phosphate-buffered saline solution (pH 7.4), as shown in. In addition, both betaine and glycerol have humectant properties, acting as a protective barrier for the skin by protecting it from water loss, allergens and bacteria, thus preserving its natural integrity. These properties are relevant to the cosmetic industry.

[0101] Example 3. Water removal by lyophilization

[0102] The water in the extract obtained in Example 2 was removed by a lyophilisation process to ensure that the stability of the mycosporines-like amino acids and, mainly phycoerythrin was preserved.

[0103] Example 4. Analysis of the extract obtained

[0104] The extraction yield is calculated as the ratio between the concentration of the compound in the sample (mycosporines-like amino acids or phycoerythrin) multiplied by the volume of solvent, and the mass of fresh biomass used. The compounds were quantified using ultraviolet-visible spectroscopy (UV / VIS) (Synergy HT-BioTek) in a range between 200-700 nm.

[0105] The quantification of mycosporines-like amino acids and R-phycoerythrin was carried out on the basis of wavelengths already described in the literature (338 nm for mycosporines-like amino acids and 565 nm for R-phycoerythrin) and in comparison with commercial compounds.

[0106] Examples of characterization of the most abundant mycosporines-like amino acids present in the biomass and the relative abundance of each of the most abundant compounds present in the sample are presented inand in Table 1. The sample was prepared by a solid-liquid extraction done to the same biomass but using only water as the solvent.

[0107]

[0108] Table 1 – Composition of the extract obtained from the same biomass but using only water as solvent in terms of mycosporines-like amino acids and their molecular ion species and fragments (m / z) data, with the relative abundance indicated for each of the mycosporines-like amino acids identified in the sample of this example, wherein a final extract has a predominant abundance of R-phycoerythrin, porphyra-334 and mycosporine-methylamine-serine.SampleRt (min)λ (nm)[M+H]+(m / z)Compound% AreaExtract ofmycosporines-like amino acids and R-phycoerythrin1.59250, 330290 (272)Mycosporine-methylamine-serine30.342.19194, 332347Porphyra-33453.9610.26208, 356216Unknown3.01

[0109]

[0110] The samples obtained in Examples 2 and 3 were subjected to four different temperatures (room temperature (20-25°C), 55°C, 75°C and 95°C) to assess their thermo-stability over time, and compared with the conventional processes, a phosphate-saline buffer solution (pH 7.4). At room temperature (20-25°C), the photo-stability of the compounds was also assessed by subjecting the extracts to light and dark conditions.

[0111] The study of the stability of the samples allows the evaluation of the half-life time (t(1 / 2)), the degradation constant (Kd) for different temperatures and the activation energy (Ea) regarding the mycosporines-like amino acids and R-phycoerythrin (Table 2), parameters determined for dark and light exposure scenarios (Table 3), parameters needed to verify the viability of the commercialization of the compounds.

[0112]

[0113] Table 2 - Thermal activation energy (Ea), thermal degradation constant (Kd) and half-life time (t(1 / 2)) data for the evaluation of the thermostability of the extracts of (A) mycosporines-like amino acids and (B) R-phycoerythrin at 55°C, 75°C and 95°C, using aqueous eutectic solvent, eutectic solvent after lyophilisation and, phosphate-buffered saline (pH 7.4), used as conventional solvent.A55°C75°C95°CEa(KJ.mol-1)Kdt(1 / 2)Kdt(1 / 2)Kdt(1 / 2)Convencional process0.0026449.700.068917.420.80481.55143.89Aqueous eutectic solvent0.0026458.870.025049.310.118810.4396.40Liophilized eutectic solvent0.0017604.510.010798.330.081812.7797.61

[0114] Kd(mL solvent.mg compound-1.h-1); t(1 / 2)(h)B55°C75°C95°CEa(KJ.mol-1)Kdt(1 / 2)Kdt(1 / 2)Kdt(1 / 2)Convencional process0.881627.18145.230.0940307.100.0447148.83Aqueous eutectic solvent1.25089.00693.530.1068216.240.0461130.85Liophilized eutectic solvent0.199965.6723.880.696749.790.3272140.3

[0115] Kd(mL solvent.mg compound-1.h-1); t(1 / 2)(h)

[0116]

[0117] Table 3 - Thermal degradation constant (Kd) and half-life time (t(1 / 2)) data for the photo-stability assessment of (A) mycosporines-like amino acid extracts and (B) R-phycoerythrin at (20-25)°C, in light and dark conditions, using aqueous eutectic solvent, eutectic solvent after lyophilisation and, as a conventional solvent, phosphate-buffered saline (pH 7.4).

[0118] A(20-25)°C(dark)25°C(light)Kdt(1 / 2)Kdt(1 / 2)Convencional process-UD-UDAqueous eutectic solvent-UD-UDLiophilized eutectic solvent-UD-UD

[0119] Kd(mL solvent.mg compound-1.h-1); t(1 / 2)(h); UD = Undetermined.

[0120] B(20-25)°C(dark)(20-25)°C(light)Kdt(1 / 2)Kdt(1 / 2)Convencional process0.0213779.390.1653102.66Aqueous eutectic solvent0.00552054.280.139687.18Liophilized eutectic solvent0.00275848.700.0204735.81

[0121] Kd(mL solvent.mg compound-1.h-1); t(1 / 2)(h).

[0122]

[0123] According to the study carried out, it was found that although the increase in temperature decreases the value of the half-life time and increases the degradation rate - Kd, the eutectic solvent betaine:glycerol, for any temperature, allows for an increase in the thermo-stability of mycosporines-like amino acids and R-phycoerythrin compared to conventional processes, such as a phosphate-salt buffer solution (pH 7.4).

[0124] According to the study carried out, it was found that at (20-25)ºC, despite the exposure of the samples to light conditions decreasing the value of the half-life time and increasing the degradation rate - Kd, the eutectic solvent betaine:glycerol allows for an increase in the photo-stability of R-phycoerythrin compared to conventional processes, such as a phosphate-salt buffer solution (pH 7.4). After 35 days, the photostability of mycosporines-like amino acids remains undetermined, which is to be expected given that only high temperatures, especially above 60ºC, promote their decomposition.

[0125] The comparison between the extracts obtained in Example 2 and Example 3 in terms of stability makes it possible to verify the need for the step of removing water from the sample, as the samples without water have the highest half-life values and the lowest degradation rate values - Kd.

[0126] The absorption of all the UV-A and UV-B radiation was confirmed by measuring the UV / VIS absorption / transmittance spectra using a Lambda 950 dual beam spectrometer (Perkin-Elmer), equipped with a 150 mm Spectralon® integrating sphere and a quartz cell with a 1 cm optical path. Measurements were made for the pure solvent (betaine:glycerol in a molar ratio of 1:2) and the extract of mycosporines-like amino acids and phycoerythrin from Example 3 obtained after 35 days of exposure to light and dark conditions at room temperature (Figures 3A and 3B).

[0127] The measurement of the pure solvent serves to confirm the attribution of the UV and HEV blue radiation filtering capacity to the compounds present in the sample.

[0128] Through the transmittance data, the theoretical sun protection factor (SPF) was calculated for the extract samples after stabilization at dark and light conditions. To note that these results were measured by using an optical path of 1 cm (Table 4).

[0129] Table 4. Theoretical SPF calculated for the final extract rich in mycosporines-like amino acids and R-phycoerythrin obtained after 35 days of exposure to light and dark conditions at room temperature. The SPF values were determined through the transmittance data obtained for an optical path of 1 cm.SampleSPFExtract ofmycosporines-like amino acids and R-phycoerythrin (20-25 ºC) dark sample1603Extract ofmycosporines-like amino acids and R-phycoerythrin (20-25 ºC) light sample524

[0130]

[0131] Using the same data of transmittance for both samples, it was possible to determine the capacity of the extract to absorb HEV blue light radiation also responsible for damaging the skin and originated by the human exposure to electronic devices. The capacity of the extract to absorb HEV blue light radiation is determined by the % HEV attenuation (higher values of this parameter indicate the high capacity of the extract to absorb the HEV radiation and protect any material from the effect of this radiation. The values obtained for both extracts are presented in Table 5.

[0132] Table 5. Percent attenuation values for HEV blue light (% HEV attenuation) at the common HEV wavelengths representing this type of radiation (415 nm, 440 nm, and 465 nm). The % HEV attenuation values were determined through the transmittance data obtained for an optical path of 1 cm.% HEV attenuationSample415 nm440 nm465 nmExtract ofmycosporines-like amino acids and R-phycoerythrin (20-25 ºC) dark sample989491Extract ofmycosporines-like amino acids and R-phycoerythrin (20-25 ºC) light sample938476

[0133]

[0134] Example 5. Cytotoxicity evaluation

[0135] Cell viability was evaluated in the human cell line of non-tumor origin HaCat (keratinocytes), by the MTT assay (thiazolyl blue tetrazolium bromide).

[0136] Cells were maintained in DMEM supplemented with 10% (v / v) fetal bovine serum, 100 U.mL-1penicillin and 0.1 mg.mL-1streptomycin. Cultures were incubated at 37°C under a humidified atmosphere containing 5% CO2.

[0137] For assays, cells were seeded in complete medium in 96-well plates. After 24h, the cultures were exposed to the samples under study, tested at five final concentrations, between 1 and 50 µg.mL-1, during an incubation period of 24 h. Afterwards, the MTT reduction test was carried out according to the procedure described in Wagemakeret al.

[0138] Three independent assays were performed, each containing four replicate cultures for each condition. As positive control, 5% (v / v) DMSO was used, which markedly reduced cell viability, as expected.

[0139] The evaluation of the cytotoxicity of the obtained extract revealed reduced cell viability in a concentration-dependent manner, as shown in. The IC50 value of the sample regarding this example was 11.9 µg.mL-1. However, it must be mentioned that the detected toxic effects were partially attributed to the intrinsic cytotoxicity of the pure solvent, which presented an IC50 value of 31.7 µg.mL-1.

[0140] As used in this description, the expressions “about” and “approximately” refer to a range in values of roughly 10% of the specified number.

[0141] As used in this description, the expression “substantially” means that the real value is within an interval of about 10% of the desired value, variable or related limit, particularly within about 5% of the desired value, variable or related limit or particularly within about 1% of the desired value, variable or related limit.

[0142] Moreover, the term “or” is intended to mean an inclusive “or” rather than an exclusive “or.” That is unless specified otherwise, or clear from the context, the phrase “X employs A or B” is intended to mean any of the natural inclusive permutations. That is, the phrase “X employs A or B” is satisfied by any of the following instances: X employs A; X employs B; or X employs both A and B.

[0143] In addition, the articles “a” and “an” as used in this application and the appended claims should generally be construed to mean “one or more” unless specified otherwise or clear from the context to be directed to a singular form.

[0144] Further, as used herein, the term “exemplary” is intended to mean serving as an illustration or example of something and is not intended to indicate a preference.

[0145] The subject matter described above is provided as an illustration of the present invention and must not be interpreted to limit it. The terminology used with the purpose of describing specific embodiments, according to the present invention, must not be interpreted to limit the invention. As used in this description, the definite and indefinite articles, in their singular form, aim to include in the interpretation the plural forms, unless the context of the description explicitly indicates the contrary. It will be understood that the expressions “comprise” and “include”, when used in this description, specify the presence of the characteristics, the elements, the components, the steps, and the related operations, but do not exclude the possibility of other characteristics, elements, components, steps, and operations from being also contemplated.

[0146] All modifications, providing that they do not modify the essential features of the following claims, must be considered within the scope of protection of the present invention.Non Patent Literature

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[0153] N. N. Rosic, C. Braun and D. Kvaskoff, in Natural Products From Marine Algae, eds. D. B. Stengel and S. Connan, Springer Science+Business Media New York, 2015, vol. 1308, pp. 119–129.

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[0157] T. A. V. Carlos, K. M. dos S. P. Cavalcante, F. de C. E. de Oliveira, C. de Ó. Pessoa, H. B. Sant’Ana, F. X. Feitosa and M. V. P. Rocha, J. Appl. Phycol., 2021, 33, 929–938.

[0158] R. D. P. Rodrigues, F. C. De Castro, R. S. De Santiago-Aguiar and M. V. P. Rocha, Algal Res., 2018, 31, 454–462.

[0159] M. Castro-Puyana, M. Herrero, J. A. Mendiola and E. Ibánez, in Functional ingredients from algae for foods and nutraceuticals, ed. H. Domínguez, Woodhead Publishing Limited, 213AD, pp. 534–560.

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Claims

A method for obtaining and stabilising mycosporines-like amino acids and phycoerythrin from biological matricescharacterized bycomprising the following steps:a) Executing at least one grinding and cell disruption step of at least one biological matrix, obtaining a ground biomass; andb) Extracting the ground biomass obtained in Step (a) with an aqueous eutectic solvent solution, obtaining an extract comprising mycosporines-like amino acids and phycoerythrin; andc) Separating a liquid phase comprising mycosporines-like amino acids and phycoerythrin from a solid phase comprising the rest of the biomass obtained in Step (b);wherein the aqueous eutectic solvent solution comprises water in a range from 5 to 95 % (w / w) in relation to the overall mass of said aqueous eutectic solvent solution; andwherein the aqueous eutectic solvent solution comprises at least a first compound and a second compound; andwherein the first compound is a hydrogen bond acceptor compound, and the second compound is a hydrogen bond donor compound.The method for obtaining and stabilising mycosporines-like amino acids and phycoerythrin, according to the previous claim,whereinthe method comprises the following further step:d) Removing substantially all the water from the liquid phase comprising mycosporines-like amino acids and phycoerythrin, obtained in Step (c), obtaining a concentrated extract comprising mycosporines-like amino acids and phycoerythrin.The method for obtaining and stabilising mycosporines-like amino acids and phycoerythrin, according to any one of the previous claims,whereinthe aqueous eutectic solvent solution comprises 10 to 30 % (w / w) of the first compound and 20 to 40 % (w / w) of the second compound.The method for obtaining and stabilising mycosporines-like amino acids and phycoerythrin, according to any one of the previous claims,whereinthe biological matrix is selected from the group consisting of an algae matrix, a fungi matrix, a bacteria matrix or mixtures thereof.The method for obtaining and stabilising mycosporines-like amino acids and phycoerythrin, according to any one of the previous claims,whereina first grinding and cell disruption step in executed in Step (a) by grinding the biological matrix with liquid nitrogen, wherein an optional second grinding step is executed after said first grinding and cell disruption step.The method for obtaining and stabilising mycosporines-like amino acids and phycoerythrin, according to any one of the previous claims,whereinthe first grinding and cell disruption with liquid nitrogen is executed in a temperature in the range of 4ºC to 40ºC.The method for obtaining and stabilising mycosporines-like amino acids and phycoerythrin, according to any one of the previous claims,whereinthe pH of the aqueous eutectic solvent solution is in the range from 5 to 8.The method for obtaining and stabilising mycosporines-like amino acids and phycoerythrin, according to any one of the previous claims,whereinthe first compound, which is comprised in the aqueous eutectic solvent solution, is selected from the group consisting of 1,2-propanediol, citric acid, decanoic acid, lauric acid, lactic acid, capric acid, malic acid, myristic acid, octanoic acid, oleic acid, palmitic acid, nonanoic acid, caffeic acid, 2,3-dihydroxysuccinic acid, alanine, arginine, betaine, fructose, L-carnitine, L-glucose, L-glutamic, nicotinamide, proline, trimethylglycine, glycerol, d-glucose, maltose, sucrose, xylose, thymol, menthol, glycine, histidine, lysine, ornithine, serine, threonine, potassium carbonate, sodium propionate, sodium acetate, choline chloride, choline acetate, acetyl tributyl citrate, triethyl citrate, tributyl citrate, L-cystine, ethylacetate, glycerol triacetate, glycerol monostearate, ethyl oleate or mixtures thereof; and the second compound, which is comprised in the aqueous eutectic solvent solution is selected from the group consisting of glycerol, betaine, ethanediol, 1,2-propanediol, 1,3-propanediol, 1,6-hexanediol, panthenol, trehalose, hexadecanoic acid, lactic acid, lauric acid, octanoic acid, oleic acid, palmitic acid, stearic acid, succinic acid, decanoic acid, nonanoic acid, pyruvic acid, acetic acid, ascorbic acid, aspartic acid, benzoic acid, citric acid, fumaric acid, leucine acid, malic acid, propionic acid, tartaric acid, caffeic acid, L-glucose, sorbitol, xylitol, mannitol, D-glucose, fructose, sucrose, alanine, arginine, cysteine, glutamic acid, glutamine, glycine, histidine, inositol, lysine, nicotinic acid, serine, proline, 2-methoxyphenol, quercetin, vanillic acid, vanillin, guaiacol, gallic acid, thereonine, menthol, thymol, leucine or mixtures thereof.The method for obtaining and stabilising mycosporines-like amino acids and phycoerythrin, according to any one of the previous claims,whereinthe molar ratio between both the first compound and the second compound in the aqueous eutectic solvent solution is in the range of 10:1 to 1:10.The method for obtaining and stabilising mycosporines-like amino acids and phycoerythrin, according to any one of the previous claims,whereinthe extraction in Step (b) is executed by mixing the biomass obtained in Step (a) with the aqueous eutectic solvent solution in a proportion in the range of 0.0001 to 0.50 grams of biomass per mL of aqueous eutectic solvent solution.The method for obtaining and stabilising mycosporines-like amino acids and phycoerythrin, according to any one of the previous claims,whereinthe extraction in Step (b) is executed in a temperature in the range of 4ºC to 50ºC.The method for obtaining and stabilising mycosporines-like amino acids and phycoerythrin, according to any one of the previous claims,whereinthe solid-liquid separation in Step (c) is at least one of the group consisting of a filtration or a centrifugation.The method for obtaining and stabilising mycosporines-like amino acids and phycoerythrin, according to any one of the previous claims,whereina lyophilisationis executed in Step (d) for removing substantially all the water from the liquid phase comprising mycosporines-like amino acids and phycoerythrin.The method for obtaining and stabilising mycosporines-like amino acids and phycoerythrin, according to any one of the previous claims,whereinthe mycosporines-like amino acid is at least one of the group consisting of shinorine, porphyra-334, palythine, palythinol, asterina-330, mycosporine-glycine, mycosporine-taurine, usujirene, mycosporine-glutaminol-glucoside , mycosporine-glutaminol, mycosporine-glutamic acid-glycine, mycosporine-glycine, mycosporine-methylamine-threonine, mycosporine-methylamine-serine, mycosporine-serinol, porphyra-331 or gadusol.The method for obtaining and stabilising mycosporines-like amino acids and phycoerythrin, according to any one of the previous claims,whereinthe phycoerythrin is R-phycoerythrin.A composition comprising mycosporines-like amino acids and phycoerythrin,characterized bybeingprepared by the method defined in any one of the previous claims.A cosmetic compositioncharacterized bycomprising thecomposition comprising mycosporines-like amino acids and phycoerythrin, as defined in the previous claim.

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