WO2025073864A1 - Mixture of esterified lignin and carboxylic acids - Google Patents

Abstract

A composition comprising esterified lignin and carboxylic acids, in particular, a mixture of a modified lignin and oleic acid, wherein the modified lignin is a lignin wherein at least one of the hydroxyl groups is esterified with oleic acid. For example, this mixture has resulted to be useful for use in the protection of a human, or animal living body, from ultraviolet radiation, protection of oxidative stress, prevention and/or treatment of acne, as well as, for increasing the pigmentation. It is also useful as preservative against microbe, and as film former. It also relates to its preparation process.

Description

Mixture of esterified lignin and carboxylic acids

This application claims the benefit of European Patent Application EP23383019 filed 04 October 2023 and of European Patent Application EP24382528 filed 16 May 2024.

Technical Field

The present invention relates to a composition comprising oleic lignin together with oleic acid as well as its uses and preparation process.

Background Art

Lignin is the most abundant polyaromatic biopolymer. Various type of lignin can be obtained depending on the biomass source and through various processes. Common types of lignin include Kraft lignin, lignosulfonates, organosolv lignin, steam exploded lignin, and enzymatic hydrolysis lignin. Regardless of the type of lignin, various substituted phenyl propane units bound to each other via aryl ether or carbon-carbon linkages.

Lignin is a promising candidate in various applications, including reduction of wettability of hydrophilic materials, addition of functionalities, uses such as protection from UV light, as antioxidant, as antimicrobial, and for tailoring of materials and formulations, e.g., for controlled substance release, adsorption, or antifouling mechanisms.

However, chemical modification of lignin is often required for most of its applications. Such modifications frequently make use of lignin's hydroxyl groups, for example, by grafting reactions during phosphorylation, sulfomethylation, esterification, or amination.

Fatty acid esterified lignin has shown to have increased non-polar solubility, reduced glass transition temperature (T_g), and increased hydrophobicity (Lewis et al. (1943) "Lignin esters of mono-and dibasic aliphatic acids," Industrial & Engineering Chemistry 35(10), 1113-1117; Pawar ef al. (2016). "Engineering physical and chemical properties of softwood kraft lignin by fatty acid substitution," Industrial Crops and Products 89, 128- 134.). Hult et al. Enhancing the barrier properties of paper board by a novel lignin coating, Ind. Crops Prod., 2013, 50, 694-700 reported that lignin palmitate and laurate derivatives have high potentials as water vapor and oxygen barrier coatings in fiber-based packaging.

W02015/094099A1 relates to a composition with a high lignin content which has been functionalized with ester groups, in a fatty acid or oil and a method of preparing said composition where the esterification step may be performed in the fatty acid or oil which is meant to be used in the production of fuels. The fatty acid used in the W02015/094099A1 , as fatty acid or as esterified fatty acid, is a C4-C14 or longer fatty acid. The oil may be tall oil, crude oil, mineral oil, and hydrocarbon oil.

While the applications of esterified lignin are promising, ongoing research is still addressing different challenges. These include process efficiency, achieving consistent properties due to the variation in the source material, compatibility with other compounds, and performance in different fields.

For the aforementioned reasons, there is still a need to improve esterified lignin derivates. Especially when some derivates underperform pure lignin in some fields. Therefore, it is desirable to develop new products based on lignin with improved properties with respect to the ones known in the art.

Summary of Invention

Inventors have found that a modified lignin, which is modified with oleic acid, in combination with free oleic acid outperforms free lignin and esterified lignin in several fields such as cosmetic UV filters, antioxidants agents, pigmentation, antimicrobial capacity which improves the preservation of cosmetic formulations, and film forming properties. In particular, the modified lignin is modified with oleic acid where at least one of the hydroxyl groups of the modified lignin has been substituted with ester groups forming esterified lignin. As far as the inventors know, there is no disclosure in the prior art relating to the combination of this modified lignin together with oleic acid. Furthermore, other modified lignins, which are modified with a (Cs-Cis) carboxylic acids, in combination with free Cs-Cis acids also have shown good antioxidant results.

Thus, in a first aspect, the present invention relates to a composition comprising a mixture of a modified lignin and a (Cs-Cis) carboxylic acid, wherein the modified lignin is a lignin wherein at least one of the hydroxyl groups is esterified with an acid selected from the group consisting of oleic acid, linoleic acid, stearic acid, 2-ethylhexanoic acid; the (Cs-Cis) carboxylic acid is selected from the group consisting of oleic acid, linoleic acid, stearic acid, 2-ethylhexanoic acid; and the modified lignin and the (Cs-Cis) carboxylic acid are in a weight ratio from 80:20 to 60:40.

In another aspect, the present invention relates to a composition as defined above, for use in the protection of a human, or animal living body, from ultraviolet radiation, wherein the composition is for topical application to the skin. It is part of the present invention the use of a composition as defined above in the protection of a human, or animal living body, from ultraviolet radiation, wherein the composition is for topical application to the hair.

In another aspect, the present invention relates to a composition as above, for use in the protection of a mammal, including a human, from oxidative stress.

In another aspect, the present invention relates to a composition as defined above, for use in preventing pigmentation in a mammal, including a human.

In another aspect, the present invention relates to the use of the composition as defined above, to improve the preservation of cosmetic formulations.

In another aspect, the present invention relates to the use of a composition as defined above as film former.

In another aspect, the present invention relates to the use of a composition as defined above, as an antioxidant.

In another aspect, the present invention relates to a composition as defined above for use in the prevention and/or treatment of acne.

Finally, in another aspect, the present invention relates to a process for preparing the composition as defined above, which comprises reacting lignin with an excess of a compound selected from oleyl halide and oleyl anhydride with respect to the hydroxyl groups of the lignin in an appropriate solvent.

Brief Description of Drawings

FIG. 1 : Comparison of the SPF values of 2% oleic lignin (Lig-syn-019), 10% oleic lignin (Lig-syn-019), and control sample.

FIG. 2: Comparison of the SPF values of 2% oleic lignin (Lig-syn-019) + 2% avobenzone, 2% avobenzone, and control and control sample.

FIG. 3: Comparison of the SPF values of 10% oleic lignin (Lig-syn-019) + 3% avobenzone, 3% avobenzone, and control and control sample.

FIG. 4: Comparison of the LIVA-PF values of 2% oleic lignin (Lig-syn-019), 10% oleic lignin (Lig-syn-019) + 3% avobenzone, 3% avobenzone, and control and control sample.

FIG. 5: Comparison of the SPF values of formulations containing pure oleic lignin (Lig- syn-030-04), oleic lignin (Lig-syn-019), and a control sample.

FIG. 6: Comparison of the LIVA-PF values of formulations containing pure oleic lignin (Lig- syn-030-04), oleic lignin (Lig-syn-019), and a control sample.

FIG. 7: Percentage of cell viability after 24 hours of exposure to different concentrations of the compounds measured by MTT assay in HaCaT cell line. Bars represent the weight average of six technical replicates, and error bars correspond to the standard deviation. Negative control (C-): cells not treated with any compound (See FIGs. 7A and 7B).

FIG. 8: Percentage of oxidative stress after cell exposure to 10 J/m² sunlight irradiation and treatment with different concentrations of the test compounds measured by in vitro H2DFFDA assay in HaCaT cell line. Bars represent the weight average of three technical replicates and error bars correspond to the standard deviation. Irradiated negative control (C- irr): irradiated cells not treated with any compound. Nonirradiated negative control (C- NO irr): nonirradiated cells not treated with any compound. Student T- test: * and #p<0.05 relative to C- Irr.

FIG. 9: Percentage of antioxidant activity after cell exposure to 10 J/m² sunlight irradiation and treatment with different concentrations of the test compounds measured by in vitro H2DFFDA assay in HaCaT cell line. Bars represent the average of three technical replicates and error bars correspond to the standard deviation. Irradiated negative control (C- irr): irradiated cells not treated with any compound. Nonirradiated negative control (C- NO irr): nonirradiated cells not treated with any compound. Student T-test: * and # p<0.05 relative to C- Irr.5.

FIG. 10: Cell viability percentage of hTERT melanocytes after a 72 hours-exposure to the indicated concentrations of the testing compounds. C- corresponds to cells without any treatment. Bars and error bars represent the mean and the standard error mean, respectively, of six technical replicates corresponding to one independent experiment. A one-way ANOVA analysis followed by a multiple comparisons test (Dunnet’s test) was performed. Statistical differences with P values <0.05 versus the negative control, C-, are represented with *; P values <0.01 versus the negative control, C-, are represented with **; P values <0.001 versus the negative control, C-, are represented with ***; and P values <0.0001 versus the negative control, C-, are represented with **** (See FIGs. 10A and 10B). FIG. 11 : Melanin production by hTERT. Cells were exposed to oleic lignin (lig-syn-019) or resveratrol 2 pg/mL for a period of 72 hours. At that time, intracellular melanin was measured and normalized to total protein concentration. Bars represent the average of at least 4 replicates and the error bars, the standard error mean. A one-way ANOVA analysis followed by a multiple comparison test (Holm-Sidak’s)) was performed. Statistical differences with P values <0.05 versus the negative control, C-, are represented with *; P values <0.01 versus the negative control, C-, are represented with **; P values <0.001 versus the negative control, C-, are represented with ***; and P values <0.0001 versus the negative control, C-, are represented with ****.

FIG. 12: Results of the challenge test (ISO 11930:2019/A1 :2022). All the tested microorganisms were evaluated at day 7, 14 and 28 after the inoculation day. N: number of microorganisms present in standardized suspensions, NO: number of microorganisms inoculated in the formula at time 0, Nx: number of surviving microorganisms, Rx: reduction of surviving microorganisms.

FIG. 13: Contact angle measurement. For each sample, 3-4 drops of 5 pL of each reagent were measured using the Sessile Drop method. The results were collected as screen captures of the drops (bottom) and in an Excel spreadsheet that specifies the left contact angle, the right contact angle, the average contact angle (top) (See FIGs. 13A and 13B).

Detailed description of the invention

All terms as used herein in this application, unless otherwise stated, shall be understood in their ordinary meaning as known in the art. Other more specific definitions for certain terms as used in the present application are as set forth below and are intended to apply uniformly through-out the specification and claims unless an otherwise expressly set out definition provides a broader definition.

For the purposes of the invention, any ranges given include both the lower and the upper endpoints of the range. Ranges given, such as temperatures, times, sizes, and the like, should be considered approximate, unless specifically stated.

The expression "pharmaceutically or cosmetically acceptable excipients or carriers" refers to pharmaceutically acceptable materials, compositions, or vehicles. Each component must be pharmaceutically or cosmetically acceptable in the sense of being compatible with the other ingredients of the pharmaceutical or cosmetical composition. It must also be suitable for use in contact with the skin of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity or other problems or complications commensurate with a reasonable benefit/risk ratio.

The term “cosmetically acceptable” which is herein used interchangeably refers to that excipients or carriers suitable for use in contact with skin or human and animals without undue toxicity, incompatibility, instability, allergic response, among others.

In the present application the term "lignin" means a polymer comprising coumaryl alcohol, coniferyl alcohol and sinapyl alcohol monomers.

In the present application the term "modified lignin" means molecules or polymers derived from lignin. In the context of the present invention, the term “modified lignin” refers to a lignin wherein at least one of the hydroxyl groups is esterified with oleic acid, i.e. , a lignin in which at least one of the hydroxyl groups is substituted with an oleyl group. This modified lignin is named herein as lignin oleate or oleic lignin indistinctly.

The term "UVA" refers to ultraviolet electromagnetic radiation with a wavelength from about 320 nm to about 400 nm.

The term "UVB" refers to ultraviolet electromagnetic radiation with a wavelength from about 290 nm to about 320 nm.

The Sun Protection Factor (SPF) refers to the ratio of minimal erythemal dose (MED) on skin protected by a sunscreen product to the minimal erythemal dose on the same unprotected skin.

The UVA protection factor (UVA-PF) means the ratio of the minimum UVA radiation dose necessary to induce persistent pigment darkening on the skin protected by a sunscreen product to the minimal UVA radiation dose necessary to induce the minimal darkening effect on the same unprotected skin.

The term “sunscreen composition” refers to a composition intended for topical application to provide protection for the skin or hair against the sunrays or other sources of UV radiation.

As mentioned above, one aspect of the present invention relates to a composition comprising a mixture of a modified lignin and a (Cs-Cis) carboxylic acid, wherein the modified lignin is a lignin wherein at least one of the hydroxyl groups is esterified with an acid selected from the group consisting of oleic acid, linoleic acid, stearic acid, 2- ethylhexanoic acid; the (Cs-Cis) carboxylic acid is selected from the group consisting of oleic acid, linoleic acid, stearic acid, 2-ethylhexanoic acid; and the modified lignin and the (C₈-Ci₈) carboxylic acid are in a weight ratio from 80:20 to 60:40. In a particular embodiment, the composition as defined above, comprising a mixture of modified lignin with oleic acid, wherein the modified lignin is a lignin wherein at least one hydroxyl group is esterified with oleic acid forming oleic lignin. Particularly, the modified lignin and the oleic acid are in a weight ratio from 80:20 to 60:40.

In order to obtain lignin, biomass may be treated in any suitable way known to a person skilled in the art. The biomass may be treated for instance in pulping processes or in organosolv processes.

Biomass includes, but is not limited to wood, fruits, vegetables, processing waste, chaff, grain, grasses, com, com husks, weeds, aquatic plants, hay, paper, paper products, recycled paper, shell, brown coal, algae, straw, bark or nut shells, lignocellulosic material, lignin, and any cellulose containing biological material or material of biological origin. In one embodiment the biomass is wood, preferably particulate wood such as saw dust or wood chips. The wood may be any kind of wood, hard or soft wood, coniferous tree or broad-leaf tree. A non-limiting list of woods would be pine, birch, spruce, maple, ash, mountain ash, redwood, alder, elm, oak, larch, yew, chestnut, olive, cypress, banyan, sycamore, cherry, apple, pear, hawthorn, magnolia, sequoia, walnut, karri, coolabah and beech.

In a particular embodiment, the lignin defined above is selected from the group consisting of Kraft lignin, lignosulfonates, organosolv lignin, steam exploded lignin, and enzymatic hydrolysis lignin. In another particular embodiment, the lignin is Kraft lignin.

Kraft lignin may be obtained by precipitation from the black liquor (side stream of the kraft process from hardwood or softwood) comprising the acidification with CO2 to precipitate lignin, followed by different washing steps with low pH solution to, finally, the filtration and drying of the Kraft Lignin.

Lignin from biorefinery may be obtained from lignocellulosic biomass in a two-stage process involving hydrolysis and removal of lignin with an organic solvent (usually a mixture of alcohol and water).

In another particular embodiment, the composition according to the present invention is that where the lignin has a total hydroxyl content from 3 to 10 mmol/g measured by ³¹ P NMR. In another particular embodiment, the composition according to the present invention is that where the Kraft lignin has a total hydroxyl content from 5 to 9 mmol/g measured by ³¹P NMR. In a particular embodiment, the weight average molecular weight of the lignin used ranges from 1 ,000 to 1 ,000,000 g/mol measured by size exclusion chromatography. In another particular embodiment, the weight average molecular weight of the lignin used ranges from 3,000 to 500,000 g/mol measured by size exclusion chromatography. In another particular embodiment, the weight average molecular weight of the lignin used ranges from 6,000 to 200,000 g/mol measured by size exclusion chromatography. In another particular embodiment, the weight average molecular weight of the lignin used ranges from 10,000 to 100,000 g/mol measured by size exclusion chromatography. In another particular embodiment, the weight average molecular weight of the lignin used ranges from 20,000 to 500,000 g/mol measured by size exclusion chromatography. In another particular embodiment, the weight average molecular weight of the lignin used ranges from 20,000 to 30,000 g/mol measured by size exclusion chromatography.

In another particular embodiment, the lignin is Kraft lignin. In a particular embodiment, the weight average molecular weight of the Kraft lignin used ranges from 1 ,000 to 100,000 g/mol measured by size exclusion chromatography. In a particular embodiment, the weight average molecular weight of the Kraft lignin used ranges from 2,000 to 50,000 g/mol measured by size exclusion chromatography. In a particular embodiment, the weight average molecular weight of the Kraft lignin used ranges from 3,000 to 25,000 g/mol measured by size exclusion chromatography.

In another particular embodiment, the composition of the invention is that where the degree of esterification of the hydroxyls groups of the modified lignin is equal to or higher than 90% measured by ³¹P NMR as disclosed in the examples. In another particular embodiment, the composition of the invention is that, where the degree of esterification of the hydroxyls groups of the modified lignin is equal to or higher than 95% measured by ³¹P NMR. In another particular embodiment, the composition of the invention is that, where the degree of esterification of the hydroxyls groups of the modified lignin is equal to or higher than 99% measured by ³¹P NMR. In another particular embodiment, the composition of the invention is that where the degree of esterification of the hydroxyl groups of the modified lignin is 100% measured by ³¹P NMR.

Oleic lignin with a lower degree of esterification than the ones disclosed above can also be used in combination with oleic acid for some of the purposes of the invention. Thus, the oleic lignin may also have a degree of esterification equal to or higher than 80%, equal to or higher than 70%, equal to or higher than 60%, equal to or higher than 50%, equal to or higher than 40%, equal to or higher than 30%, or equal to or higher than 20%, being the degree of esterification measured by ³¹ P NMR. In another particular embodiment, the composition of the invention is that in which the modified lignin has a weight average molecular weight from 2,000 to 400,000 g/mol measured by size exclusion chromatography as shown in the examples. In another particular embodiment, the composition as defined above, where the modified lignin has a weight average molecular a weight average molecular weight from 4,000 to 200,000 g/mol measured by size exclusion chromatography.

In another particular embodiment, the modified lignin is made from Kraft lignin. In another particular embodiment, the composition of the invention is that in which the modified lignin has a weight average molecular weight from 2,000 to 200,000 g/mol measured by size exclusion chromatography as shown in the examples. In another particular embodiment, the composition of the invention is that in which the modified lignin has a weight average molecular weight from 2,000 to 20,000 g/mol measured by size exclusion chromatography as shown in the examples. In another particular embodiment, the composition of the invention is that in which the modified lignin has a weight average molecular weight from 2,000 to 10,000 g/mol measured by size exclusion chromatography as shown in the examples. In another particular embodiment, the composition of the invention is that in which the modified lignin has a weight average molecular weight from 3,000 to 8,000 g/mol measured by size exclusion chromatography as shown in the examples.

In another particular embodiment, the composition as defined above, where the modified lignin and the oleic acid are in weight ratio from 95:5 to 40:60. In another particular embodiment, the composition as defined above, where the modified lignin and the oleic acid are in a weight ratio from 80:20 to 50:50. In another particular embodiment, the composition as defined above, where the modified lignin and the oleic acid are in a weight ratio from 75:25 to 60:40. In another particular embodiment, the composition as defined above, where the modified lignin and oleic acid are in a weight ratio of 70:30. The amount of oleic acid in the mixture can be determined by ¹H RMN.

In another particular embodiment, the composition as defined above is that where the mixture of a modified lignin and a (Cs-Cis) carboxylic acid is that wherein the modified lignin is a lignin wherein at least one of the hydroxyl groups is esterified with an acid selected from the group consisting of oleic acid, linoleic acid, and stearic acid; and the (C₈-Ci₈) carboxylic acid is selected from the group consisting of oleic acid, linoleic acid, and stearic acid.

In another particular embodiment, the composition as defined above is that where the mixture of a modified lignin and a (Cs-Cis) carboxylic acid is selected from the following ones: a) linoleate lignin and linoleic acid; b) oleate lignin and Linoleic acid; c) stereate lignin and stearic acid; d) stereate lignin and oleic acid; e) stereate lignin and 2-ethylhexanoic acid; and f) ethlyhexanoate lignin and 2-ethylhexanoic acid.

In another particular embodiment, in combination with any of the embodiments above or below, the composition as defined above, which is a selected from the group consisting of: a cosmetic composition, a pharmaceutical composition, and a personal care composition.

In another particular embodiment, the composition is a pharmaceutical, cosmetical, or personal care composition, where the mixture of modified lignin and oleic acid are in an amount from 0.1 to 30% by weight of the composition; the composition further comprises one or more pharmaceutically or cosmetically acceptable excipients or carriers, and the sum of the components being 100% by weight.

Appropriate excipients or carriers for the composition of the present invention are for instance, emollients, dispersing agents, preservatives, film-forming agents, anti-foams, perfumes, fragrances, oils, propellants, dyes, pigments, colorants, opacifiers, emulsifiers, surfactants, thickeners, humectants, antioxidants, chelating agents, pH regulating agents, solubilizers, photo stabilizers, waxes, gelifiers and exfoliants.

The compositions of the present invention can be produced by conventional processes known in the art, such as for instance by mixing the different components of the composition, in any order. The appropriate excipients and/or carriers, and their amounts, can readily be determined by those skilled in the art according to the type of the composition being prepared.

As it is illustrated in the Examples, the composition comprising oleic lignin and oleic acid shows protection against UV radiation, either alone or in combination with other filters.

Thus, it is part of the present invention a composition as defined above, for use in the protection of a human or animal living body, from ultraviolet radiation.

In a particular embodiment, in combination with any of the embodiments above or below, the composition of the present invention for use as defined above, is that where the protection is for the UV radiation emitted by the sun. In a particular embodiment, the composition for use as defined above, further comprises a UV sunscreen agent.

In another particular embodiment, in combination with any of the embodiments above or below, the composition for the use as defined above is a sunscreen composition. A sunscreen composition comprising the composition defined above or below is also part of the present invention.

The term “sunscreen active agent”, as used herein, relates to materials, singly or in combination, that are regarded as acceptable for use as active sun screening ingredients relative to their ability to absorb, scatter, or reflect UV radiation. Such compounds are generally described as being UVA, UVB, or UVA/UVB sunscreen active agents, depending on the wavelength range of the UV radiation.

Examples of UV sunscreen active agents that can be included in the composition are the following: octyl salicylate (2-ethylhexyl salicylate, Escalol 587); pentyl dimethyl PABA; octyl dimethyl PABA (padimate O, Escalol 507); benzophenone-1 ; benzophenone-6 (Uvinul D-49); 2-(2H-benzotriazole-2-yl)-4,6-di-tert-pentylphenol (Uvinul 3028); ethyl-2- cyano-3,3-diphenylacrylate (Uvinul 3035); homomenthyl salicylate (homosalate); bisethylhexyloxyphenol methoxyphenyl triazine (bemotrizinol, Escalol S); methyl-(1 ,2,2,6,6- pentamethyl-4-piperidyl)-sebacate (Uvinul 4092H); benzenepropanoic acid, 3,5-bis (1,1- dimethyl-ethyl)-4-hydroxy-, C7-C9 branched alkyl esters (Irganox 1135); 2-(2H- benzotriazole-2-yl)-4-methylphenol (Uvinul 3033P); diethylhexyl butamido triazone (iscotrizinol, Uvasorb HEB); amyl dimethyl PABA (lisadimate, glyceryl PABA); 4,6- bis(octylthiomethyl)-o-cresol (Irganox 1520); CAS number 65447-77-0 (Uvinul 5062H, Uvinul 5062GR); red petroleum; ethylhexyl triazone (Uvinul T-150); octocrylene (Escalol 597); isoamyl-p-methoxycinnamate (amiloxate, Neo Heliopan E1000); drometrizole; titanium dioxide (including as nanomaterial); 2,4-di-tert-butyl-6-(5-chloro-2H-benzotriazole- 2-yl)-phenol (Uvinul 3027); 2-hydroxy-4-octyloxybenzophenone (Uvinul 3008); benzophenone-2 (Uvinul D-50); diisopropyl methylcinnamate; PEG-25 PABA; 2-(1 ,1- dimethylethyl)-6-[[3-(1 , 1 -demethylethyl)-2-hydroxy-5-methylphenyl]methyl-4-methylphenyl acrylate (Irganox 3052); drometrizole trisiloxane (Mexoryl XL); menthyl anthranilate (meradimate); bis-(1 ,2,2,6,6-pentamethyl-4-piperidyl)-sebacate; butyl methoxydibenzoylmethane (avobenzone, Escalol 517); 2-ethoxyethyl p- methoxycinnamate (1 lolyacry); benzylidene camphor sulfonic acid (Mexoryl SL); dimethoxyphenyl-[1-(3,4)]-4,4-dimethyl-1,3-pentanedione; zinc oxide (including as nanomaterial); N,N’-hexane-1,6-diyl-bis [3-(3,5-di-tert-butyl-4- hydroxyphenylpropionamide)] (Irganox 1098); pentaerythritol tetrakis[3-(3,5-di-tert-butyl-4- hydroxyphenyl)propionate] (Irganox 1010); 2,6-di-tert-butyl-4-[4,6-bis(octylthio)-1 ,3,5- triaziN-2-ylamino] phenol (Irganox 565); 2-(2H-benzotriazole-2-yl)-4,6-bis(1-methyl-1- phenylethyl)phenol (llvinul 3034); trolamine salicylate (triethanolamine salicylate); diethylanolamine p-methoxycinnamate (DEA methoxycinnamate); polysilicone-15 (Parsol SLX); CAS number 152261-33-1 (llvinul 5050H); 4-methylbenzylidene camphor (Eusolex 6300, Parsol 5000); bisoctrizole (Tinosorb M), including as nanomaterial; benzenamine, N-phenyl-, reaction products with 2,4,4-trimethylpentene (Irganox 50507); sulisobenzone, Escalol 577); (2-ethylhexyl)-2-cyano-3,3-diphenylacrylate (Uvinul 3039); digalloyl trioleate; 12olyacrylamide methylbenzylidene camphor; glyceryl ethylhexanoate dimethoxycinnamate; 1 ,3-bis-[(2’-cyano-3’,3’- diphenylacryloyl)oxy]-2,2-bis-{[(2’-cyano-; bis-(2,2,6,6-tetramethyl-4-piperidyl)-sebacate (Uvinul 4077H); benzophenone-5 (sulisobenzone sodium); 1 ,3,5-tris(3,5-di-tert-butyl-4- hydroxybenzyl)-1 ,3,5-triazine- 2,4,6(IH,3H,5H)-trione (Irganox 3114); 1,3,5-Triazine, 2,4,6-tris(1,1’-biphenyl)-4-yl-, including as nanomaterial; hexamethylendiamine (Uvinul 4050H); benzophenone-8 (dioxybenzone); ethyl-4- bis(hydroxypropyl) aminobenzoate (roxadimate); 6-tert-butyl-2- (5-chloro-2H-benzotriazole- 2-yl)-4-methylphenol (Uvinul 3026); p-aminobenzoic acid (PABA); 3,3’,3”,5,5’,5”-hexa-tert-butyl-a-a’-a”-(mesitylene-2,4,6-triyl)tri-p-cresol (Irganox 1130); lawsone with dihydroxyacetone; benzophenone-9 (Uvinul DS-49); benzophenone-4 (sulisobenzone); ethylhexyl dimethoxy benzylidene dioxoimidazoline propionate; N,N’- bisformyl-N,N’-bis- (2,2,6,6-tetramethyl-4-piperidinyl)-; 3-benzylidene camphor (Mexoryl SD); terephthalylidene dicamphor sulfonic acid; camphor benzalkonium methosulfate (Mexoryl SO); bisdisulizole disodium (Neo Heliopan AP); etocrylene; ferulic acid; 2-(2H- benzotriazole-2-yl)-4-(1 ,1,3,3-tetramethylbutyl)-phenol (Uvinul 3029); ecamsule (Mexoryl SX); 4,6-to(dodecylthiomethyl)-o-cresol (Irganox 1726); beta-2-glucopyranoxy propyl hydroxy benzophenone; phenylbenzimidazole sulfonic acid (ensulizole, Eusolex 232, Parsol HS); benzophenone-3 (oxybenzone, Escalol 567); diethylamine hydroxybenzoyl hexylbenzoate (Uvinul A Plus); 3’,3’-diphenylacryloyl)oxy]methyl}-propane (Uvinul 3030); 3,3#-(1 ,4-phenylene)bis(5,6-diphenyl-1 ,2,4-triazine); 2-ethoxyethyl (2Z)-2-cyano-2-[3-(3- methoxypropylamino) cyclohex-2-en-1-ylidene]acetate and ethylhexyl p- methoxycinnamate (Escalol 557).

The UV sunscreen active agent can be a UVB sunscreen active agent, a UVA sunscreen active agent, a UVA/UVB sunscreen active agent, or a mixture thereof. It is generally desirable to formulate sunscreen compositions with more than one UV sunscreen active agent, particularly to extend the range of UV radiation absorption provided, more particularly, to cover a broad protection against both UVA and UVB radiation.

In another particular embodiment, the composition for use as defined above, where the further UV sunscreen agent is selected from the group consisting of abovenzone, Octocrylene, Ethylhexyl salycilate, Homosalate, Bis-Ethylhexyloxyphenol Methoxyphenyl Triazine, Ethylhexyl Triazone, Diethylamino hydroxybenzoyl hexyl benzoate, Methylene Bis-Benzotriazolyl Tetramethylbutylphenol, Phenylbenzimidazole Sulfonic Acid, Butylmethoxydibenzoylmethane and Diethylhexyl Butamido Triazone, and a mixture thereof in an amount from 0.1 to 50 wt.% based on the total weight of the composition. In another particular embodiment, the UV sunscreen agent is in an amount from 0.5 to 45 wt.% based on the total weight of the composition. In another particular embodiment, the UV sunscreen agent is in an amount from 1 to 35 wt.% based on the total weight of the composition.

In another particular embodiment, in combination with any of the embodiments above or below, the cosmetic or pharmaceutical composition, or a personal care composition for use as defined above is that where the composition is selected from the group consisting of creams, ointments, oils, lotions, gels, sticks, foams, milks, suspensions, powders, emulsions, dispersions, sprays, aerosols, lipsticks, foundations, makeup, lose or press powders, eye blushes, eye shadows, mascaras, nail varnishes, nail lacquers and nonpermanent dyeing composition for the hair.

In another particular embodiment, in combination with any of the embodiments above or below, the cosmetic or pharmaceutical composition, or a personal care composition for use as defined above are for topical administration.

As illustrated in the examples, the composition comprising oleic lignin and oleic acid shows very high antioxidant activity reducing oxidative stress compared to the control sample. Due to low cytotoxicity compared to other antioxidants such as ascorbic, higher concentrations can be used, providing better antioxidant activity.

Oxidative stress refers to a phenomenon caused by an imbalance between production and accumulation of oxygen reactive species (ROS) in cells and tissues and the ability of a biological system to detoxify these reactive products. ROS can play, and in fact they do it, several physiological roles (i.e. , cell signaling), and they are normally generated as byproducts of oxygen metabolism; despite this, environmental stressors (i.e., UV, ionizing radiations, pollutants, and heavy metals) and xenobiotics (i.e., antiblastic drugs) contribute to greatly increase ROS production, therefore causing the imbalance that leads to cell and tissue damage (oxidative stress).

The antioxidant property of lignin is dependent on the phenolic hydroxy groups capable of free-radical scavenging. Lignin-derived phenols need to be lipophilized to ensure good compatibility. This can be achieved by esterifying aliphatic chains into some of the hydroxyl groups, preferably preserving the aromatic -OHs responsible for their antioxidant functionality. The development of lignin derivates synthesising techniques has improved the accessibility of the enhanced antioxidant activity compared to native lignin.

Accordingly, a composition as defined above, for use in the protection of a mammal, including a human, from oxidative stress is also part of the invention.

Examples of the present invention also illustrates that the composition of the present invention affects the pigmentation. In particular, it affects the melanin synthesis by hTERT melanocytes, producing an increase in the melanin/protein ratio.

Thus, it is also part of the present invention the use of a composition as defined above for increasing pigmentation in a mammal, including a human.

In a particular embodiment, the use of the composition as defined above for increasing pigmentation, where the increase in pigmentation is produced by increasing melanin production.

As well as the antioxidant effect, the antimicrobial effect of lignin and derivates thereof is also caused by the phenolic compounds.

In addition, the composition of the present invention has also proven to be a useful as a preservative by showing good antimicrobial properties.

Thus, it is also part of the present invention, the use of the composition as defined above, to improve the preservation of cosmetic formulations.

The composition of the present invention has also shown good properties as film former. So, it is also part of the invention the use of the composition as defined above, as a film former.

It is also part of the present invention, the use of a composition as defined above, as an antioxidant.

The compositions described in this invention are beneficial for enhancing the quality of acne-prone skin. Additionally, the product fortifies the skin's barrier function, diminishes erythema, reduces sebum production, and lessens the occurrence of blackheads and large nodules (over 5 mm in width), which are painful lumps that do not contain pus.

This composition also proactively prevents the onset of new acne breakouts and blemishes. The formula targets the underlying causes of acne, effectively preventing the formation of new lesions and skin imperfections. With regular use, it promotes and maintains clearer, healthier-looking skin by pre-emptively addressing factors that lead to acne, thereby stopping new breakouts and spots before they manifest.

Accordingly, it is also part of the invention, a composition as defined in above for use in the prevention and/or treatment of acne.

The compositions of the present invention may be prepared by a process which comprises reacting lignin with an excess of oleyl halide with respect to the hydroxyl groups of the lignin in an appropriate solvent. In another particular embodiment, the process as defined above, where the oleyl halide is oleyl chloride. In another particular embodiment, the process as defined above, where the oleyl halide is in an amount from 1.1 to 1.3 equivalents with respect to the hydroxyl groups of the lignin.

In another particular embodiment, the process as defined above, is that which comprises reacting lignin with an excess of oleyl anhydride with respect to the hydroxyl groups of the lignin in an appropriate solvent. In another particular embodiment, the process as defined above, where the oleyl anhydride is in an amount from 1.1 to 1.3 equivalents with respect to the hydroxyl groups of the lignin.

In another particular embodiment, the process as defined above, is that which is conducted at a temperature from 0 to 35 °C.

Oleic lignin with different degrees of esterification may be prepared by a process which comprises reacting lignin with oleyl halide wherein the oleyl halide is the limiting reactant, thus allowing to obtain oleic lignin with different degrees of esterification. The reaction may be conducted at a temperature from 0 to 35 °C. The composition of the present invention can be obtained by further mixing this oleic lignin with different degrees of esterification with oleic acid to obtain the composition of the present invention.

All the particular embodiments indicated above for the composition, are also particular embodiments for the process for preparing the composition.

Finally, a composition comprising a mixture of a modified lignin and oleic acid, wherein the modified lignin is a lignin wherein at least one of the hydroxyl groups is esterified with oleic acid obtainable by any of the processes defined above including any particular of the embodiments of the process is part of the invention.

Throughout the description and claims the word "comprise" and variations of the word, are not intended to exclude other technical features, additives, components, or steps. Furthermore, the word “comprise” encompasses the case of “consisting of”. Additional objects, advantages and features of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention. The following examples and drawings are provided by way of illustration, and they are not intended to be limiting of the present invention. Reference signs related to drawings and placed in parentheses in a claim, are solely for attempting to increase the intelligibility of the claim and shall not be construed as limiting the scope of the claim. Furthermore, the present invention covers all possible combinations of particular and preferred embodiments described herein.

Examples

Size Exclusion

(GPC) Samples: Concentration: 3 mg/ml Filter:

Nonsterile Nylon Syringe, 0.22 pm pore. Chromatographic conditions: Instrument: Agilent Technologies 1260 Infinity; Columns: PLgel 5 pm Guard/50 x 7.5 mm, PLgel 5 pm 1000 A MW 4K-400K and PL; Mixed gel C; Mobile phase: Tetrahydrofuran; Flowrate: 1.0 ml/min;

Injection volume: 20 pl; Temperature: 23 °C; Detector: RID A, Refractive Index Signal;

Duration: 30 min.

Infrared

: Model Bruker Alpha II; Accessory: Platinum ATR; Number of sample scans: 16; Number of background scans: 8; Resolution: 4 cm

³¹P Nuclear Magnetic Resonance (NMR)

: Instrument: Bruker BioSpin

GmbH. Avance Pulse sequence: zg; Solvent: CDCI3: Pyridine (1 :1.6 v/v); Temperature: 298 K; Scans: 170; Relaxation delay: 15 s; Pulse width: 12; Acquisition time: 0.6750 s;

Width: 48543.7; Spectrometer Frequency: 121.51 ; FT size (Spectral Size): 65536. is: Initial temperature: 30 °C Final temperature: 800 °C; lank.

ument: Eager 200; Scale: MX5 Mettler Toledo; Combustion temperature: 1200 °C; Atmosphere: Oxygen; Standard: Sulfanilamide STD; Method:

220308 N, C, H i S

¹H Nuclear Magnetic Resonance spectroscopy

Instrument: Bruker BioSpin GmbH. Avance; Pulse sequence: zg30; Solvent: CDCh Temperature: 298 K; Scans: 32; Relaxation delay: 1 - 5 s; Pulse width: 8; Acquisition time: 2.6241 ; Width: 6250.0; Spectrometer Frequency: 400.20; FT size (Spectral Size): 65536 Example 1 : Characterisation of starting material (Kraft Lignin)

Kraft lignin from softwood was used for the synthesis of the oleic lignin and was characterized using the technigues disclosed above.

Size Exclusion Chromatography (GPC: The raw material only shows one peak by GPC. This is typical of lignin, as its chains are too similar among them to be differentiated using this analytical method. This method is not precise as the standards to calibrate the instruments are not lignin-based. The molecular weight measured with this instrument is 3,231 g/mol. (Table 1).

Table 1 : Integration of the Kraft lignin peak

Infrared Spectroscopy: Infrared analysis was done of the sample as received. A band at 3,000 cm^-1 indicates the presence of free hydroxyl moieties and it is characteristic for polymeric OH groups. This band and the absence of one at 1 ,700 cm^-1 allows to identify the esterified lignin.

³¹P Nuclear Magnetic Resonance (NMR) spectroscopy: The ³¹ P NMR spectroscopy was designed to guantify the amount of hydroxyl groups in the compound and differentiate them. The free hydroxyl groups react with a phosphorus reagent and can be detected by NMR.

The ³¹ P NMR spectroscopy allows guantifying the amount of hydroxyl groups in the compound and differentiate them. The free hydroxyl groups react with a phosphorus reagent that can be detected by NMR.

A detailed description of the method to be used to guantify the hydroxyl groups by ³¹ P NMR is disclosed in Xianzhi Meng et al., “Determination of hydroxyl groups in biorefinery resources via guantitative ³¹ P NMR spectroscopy”, Nature Protocols, vol. 14, pp. 2627- 2647.

Table 2: Assigned sub-structures of Kraft lignin by ³¹P NMR

This analysis shows that most free hydroxyls are aromatic, but that appear as three different structures. Aliphatic alcohols account for 46% while the Guaiacyl moieties only for 31%. The ratios are similar to the ones reported by the manufacturer. However, the amounts (as mmol/g) are different.

Thermoqravimetry Analysis:

Table 3: Thermoqravimetry results of Kraft lignin.

The Thermogravimetry Analysis shows that Kraft lignin contains around 5% of water that is loss when heating up to 114 °C. From then, there is a constant loss of mass from degradation. The total mass loss accounts for 61.2%.

Elemental Analysis:

Table 4: Elemental analysis of Kraft lignin

Example 2. Synthesis of Oleic lignin - Characterisation

Oleic lignin refers to the product obtained in the synthesis as a mixture of oleic lignin pure and oleic acid.

Lignin (10 g) was first dissolved in THF (100 ml) and transferred to a round-bottom flask. Triethylamine (30 ml, 3.3 to 5 equiv. respect the hydroxyl groups) was added relatively fast. A solution of oleoyl chloride (25 g, 1.1 to 1.3 equiv. respect the hydroxyl groups) was dissolved in THF (50 ml) and added dropwise. The mixture was left stirring at room temperature for a minimum of two hours up to overnight. Once the reaction was finished, it was quenched with a cold aqueous solution of HCI (2-12%) by either adding it to the mixture or dropping the reaction into the acid. Two phases were formed and separated. The aqueous phase was washed three times with ethyl acetate or heptane. The organic fractions combined were washed with water and brine, dried over magnesium sulphate, filtered, and concentrated under vacuum at 40 °C. The procedure yielded a mixture of oleic lignin and oleic acid at an approximate 70:30 ratio. The ratio was calculated as follows: A known aliquot of the lignin was washed several times (3-9) with ethanol. The remaining solid and the ethanolic phase were dried separately and weighted. The ratio was calculated by dividing the weight of the ethanolic phase by the sum of weights.

Characterization

Table 5: Individual integration of the peaks of the mixture of oleic lignin (Liq-syn-050) obtained by Size Exclusion Chromatography (GPC):

After separating the oleic lignin from the oleic acid with the ethanol washes (see procedure above), the GPC chromatogram of the oleic showed only one peak, while the chromatogram of the oleic acid showed two to three peaks, being Peak 2 the most intense one. Peak 1 refers to the modified lignin. Peaks 2 and 3 refers to oleic acid.

Infrared Spectroscopy: The initial characterisation is carried out by Infrared Spectroscopy. This technique shows if the hydroxyl groups have been esterified by the disappearance of the broad band at 3,500-3,000 cm^-1. It also shows whether the sample contains carbonyl groups. The spectra of oleic lignin pure and oleic lignin were respectively conducted. The characteristic sharp bands at 2,922 and 2,852 cm^-1 seen corresponded to the CH2 groups of the oleic moiety. The carbonyl groups appeared at 1 ,700 cm^-1. The spectrums were intrinsically different to that of Kraft lignin.

¹H Nuclear Magnetic Resonance (NMR) was used to see if the product was completely dry and to detect the presence of oleic acid. The spectrum of oleic lignin pure showed broad and not defined peaks (1 H NMR: 7.19-6.48, 5.30, 4.00-3.30, 2.85-2.12, 2.00, 1.85- 1.49, 1.25, 0.88). The oleic acid spectrum matched that of the same compound on databases. The spectrum of oleic lignin overlapped with that of oleic acid indicating, that the product contained this compound. ³¹P Nuclear Magnetic Resonance (NMR) was used to see if the product had any hydroxyls groups left. The results showed that there are no free hydroxyls, therefore the esterification was complete according to this technique.

3. Formulation results

Oleic lignin refers to the product obtained in the synthesis as a mixture of oleic lignin pure and oleic acid (see example 2). Oleic lignin pure (used as comparative example) refers to Oleic lignin without oleic acid.

The first formulation tests were carried out with oleic lignin (mixture of oleic lignin pure and oleic acid, Lig-Syn-019). This oleic lignin is soluble in emollients of different polarities and at different concentrations without the need to heat the mixture.

Three formulations were carried out to see the behaviour of oleic lignin (Lig-Syn-019) alone and in combination with another commercial cosmetic UV filters and the Roka SMART UV® PvB 360 active from Roka Furadada:

• Placebo formula (without lignin)

• Formula with 2% of oleic lignin (Lig-Syn-019)

• Formula with 2% oleic lignin (Lig-Syn-019) and 2% PvB 360

• Formula with 10% of oleic lignin (Lig-Syn-019)

• Formula with 10% of oleic lignin (Lig-Syn-019) and 3% of Avobenzone

Photoactivation of these formulations was performed to see the absorbance spectrum in each case, and the results showed that the formulation that contain a 2% of oleic lignin (mixture of oleic lignin pure and oleic acid, Lig-Syn-019) alone provides a very subtle increase in SPF values compared to the placebo formulation, specifically 0.32 points. By increasing the oleic lignin (Lig-Syn-019) concentration from 2% to 10%, an increasement in the initial SPF value of 1.68 points was observed (see FIG. 1).

Moreover, in the combination of oleic lignin (Lig-Syn-019) and Roka SMART UV® PvB 360 (2% of the sunscreen agent Avobenzone), it was observed that the initial SPF value is higher when lignin is present (compared to the same formula containing only Roka SMART UV® PvB 360 (2% of the sunscreen agent Avobenzone)). As for the stability of the formulations, it was observed that the formula containing only oleic lignin (Lig-Syn-019) darkens with temperature (at 40°C), while the one containing oleic lignin (Lig-Syn-019) and Roka SMART UV® PvB 360 the same colour as at the beginning (see FIG. 2 and FIG. 3). For the combination with a commercial filter (UVA), in this case Avobenzone, it is observed that it increases the initial SPF value by 0.7 points (although it damages the initial LIVA-PF values provided by Avobenzone, as oleic lignin (Lig-Syn-019) does not provide UVA protection). Oleic lignin (Lig-Syn-019) does not influence the stability of the formula in this case (see FIG. 4).

Comparative product: pure oleic lignin (Lig-Syn-030-04), was also tested (see FIG. 5). It was more difficult to incorporate into a formula because it was more difficult to dissolve in emollients.

An SPF 20 formulation was made with commercial UV filters to test the ability of oleic lignin (Lig-Syn-019) and pure oleic lignin (Lig-Syn-030-04) to act as SPF or UVA-PF booster. The formulations tested were as follows:

• Placebo formula: Octocrylene (7.5%), Avobenzone (1.3%) and EthylhexylTriazone (2.5%).

• Formula with oleic lignin (Lig-Syn-019): Octocrylene (7.5%), Avobenzone (1.3%) and Ethylhexyl Triazone (2.5%) + 3% oleic lignin (Lig-Syn-019).

• Comparative Formula with oleic lignin pure (Lig-Syn-030-04): Octocrylene (7.5%), Avobenzone (1.3%) and Ethylhexyl Triazone (2.5%) + 3% oleic lignin (Lig-Syn030- 04).

The results of the SPF values showed that oleic lignin (Lig-Syn-019) contributed a 29% increase from the initial value. Moreover, as the product was irradiated, the value increased. Comparative Pure oleic lignin (Lig-Syn-030-04) did not contribute anything, even affecting the initial SPF value (see FIG. 6).

In the case of UVA-PF values, the formula containing oleic lignin (Lig-Syn-019) maintained the initial UVA-PF value of the placebo formula, but as the sample was irradiated, the placebo formula lost UVA protection while the one containing oleic lignin (Lig-Syn-019) maintained and even increased the protection values. The formula containing comparative pure oleic lignin (Lig-Syn-030-04), again, did not contribute anything to the UVA-PF values, it even has a negative effect on them (see FIG. 7).

Example 4. Antioxidant Activity

In the first place, a cytotoxicity study (MTT assay) was carried out to determine cell viability after exposure to different concentrations of the oleic lignin (lig-syn-019) in immortalized human keratinocytes (HaCaT cell line) and compare the results with ascorbic acid. The oleic lignin (lig-syn-019) does not show cell cytotoxicity at the concentrations tested, maintaining a cell viability above 80% in all cases. The slight reduction in cell viability at 60 pg/ml concentration is due to the solvent, DMSO, present at 1% concentration in this condition. In comparison to the oleic lignin (lig-syn-019), ascorbic acid displays higher cytotoxicity, showing subtoxic conditions below 11.1 pg/ml.

Next, to assess the antioxidant activity of the compound in comparison with ascorbic acid, an in vitro H2DFFDA assay for reactive oxygen species (ROS) detection was carried out in HaCaT cell line. Test concentrations were selected taking into consideration the results of the previous cytotoxicity study. Cells treated with the test compounds were exposed to 10 J/m² of sunlight. The results showed that oleic lignin (lig-syn-019) exhibited very high antioxidant activity at concentrations ranging from 33.33 to 0.41 pg/ml, reducing oxidative stress (measured as ROS production) compared to the non-irradiated control (C- NO irr.) level, similarly to the ascorbic acid. At lower concentrations (0.14 and 0.05 pg/ml) oleic lignin (lig-syn-019) decreased its antioxidant activity, while ascorbic acid maintained its activity. At the higher concentration tested (60 pg/ml, oleic lignin (lig-syn-019) reduced its antioxidant capacity, probably due to the cytotoxic effect of the solvent (DMSO), present at 1% concentration in this condition (see FIG. 8 and FIG. 9).

Example 5. Pigmentation

The effect of oleic lignin (lig-syn-019) with resveratrol on the modulation of melanin production by hTERT immortalized dermal melanocytes from human skin was quantified.

In the first place, cytotoxicity assay (MTT assay) was carried out on hTERT melanocytes for oleic lignin (lig-syn-019) and resveratrol at eight different concentrations after 72 hours of exposure. The oleic lignin (lig-syn-019), at concentrations ranging from 12.5 to 0.78 pg/mL, did not exhibited cytotoxic effects on hTERT melanocytes after 72 hours of exposure. Resveratrol compromised hTERT melanocytes cell viability in a dose dependent manner at all concentrations assayed, ranging from 100 pg/mL to 0.78 pg/mL (see FIG. 10).

Next, a melanin production assay on hTERT melanocytes for oleic lignin (lig-syn-019) and resveratrol at different concentrations after 72 hours of exposure by melanin quantification was conducted. Oleic lignin (lig-syn-019) followed a similar trend to resveratrol in the effect on melanin synthesis by hTERT melanocytes. The concentration of 10 pg/mL for the oleic lignin (lig-syn-019) and resveratrol increased the melanin/protein ratio over the negative control (C-). However, in the case of resveratrol, this concentration was cytotoxic, so it was excluded from all groups in the final analysis (see FIG. 11). Example 6. Preservative

The antimicrobial protection of the oleic lignin pure (Exp-LIG-FOR-008009) was evaluated performing Preservative efficacy testing (PET), also known as preservative Challenge Test (ISO 11930:2019/A1 :2022).

This test determines the effectiveness of a preservative during its shelf life and evaluates how well a product withstands microbial contamination during use. For example, in sterile vials containing multiple doses, antimicrobial preservatives are used to inhibit the growth of any microorganisms that may be introduced during repeated insertion and withdrawal to load individual amounts.

Protocol:

The product was separated out into 5 containers, each being challenged with one of the 5 method-specified microorganisms at a concentration of >1 x105 CFU/g or ml.

The initial concentration of each microorganism was determined by standard dilution and plating techniques.

At the time of test initiation, a separate aliquot of the product, typically 1 g or 1 ml, was placed in a volume of neutralization broth to be used in the neutralization and recovery validation.

The product was held at room temperature for a period of no less than 28 days.

The inoculated product was evaluated at 7, 14 and 28 days after inoculation and surviving microorganisms were enumerated using standard dilution and plating techniques.

Test sample colonies were counted at each specified interval to determine the amount of microorganisms remaining.

The log reduction of each microorganism at each interval was calculated and reported. The effectiveness of the preservative system was determined by comparison to the acceptance criteria.

Criteria:

• Criteria A: o For bacteria, the product must demonstrate no less than a 3 log reduction from the initial count at 7 days, and no increase from the 7 day count at days 14 and 28. o C. albicans must be reduced by no less than 1 log at 7 days and show no increase from the 7 day count at days 14 and 28. o A. brasiliensis must show no increase from the initial count at 14 days and no less than a 1 log reduction at 28 days.

• Criteria B: o For bacteria, the product must demonstrate no less than a 3-log reduction from the initial count at 14 days and no increase from the 14 day count at day 28. o C. albicans must be reduced by no less than 1 log at day 14 and show no increase from the 14 day count at day 28. o A. brasiliensis must show no increase from the initial count at 14 and 28 days.

The results showed that the oleic lignin pure formulation fulfilled with the evaluation criteria B which means that the product was protected if the risk analysis demonstrated the existence of control elements not related to the product, indicating that the microbiological risk was acceptable (see FIG. 12).

Example 7. Film former

The film former capacity was evaluated using a static (sessile drop) contact angle. This parameter was measured when the droplet is sitting on the surface.

In this experiment the contact angle of oleic lignin pure (Exp-LIG-FGR-008-009), oleic lignin (Exp-LIG-FGR-008-012) and a formulation with a commercial film former was compared. The results showed that both oleic lignin pure (Exp-LIG-FGR-008-009) and oleic lignin (Exp-LIG-FGR-008-012) had a better film-former capacity compared to the commercial one (see FIG. 13).

Example 8. Improve the quality of acne-prone skin

The aim of the study was to determine the anti-acne efficacy of the mix of oleic lignin and oleic acid (Lig-Syn-019), on volunteers with oily skin with mild-moderate acne tendency on a minimum of 20 volunteers, using the product for 28 days under the normal conditions.

To evaluate the efficacy of the Lig-Syn-019 on acne-prone skin, several biometric parameters were analysed, and a visual dermatological performed by a qualified dermatologist was carried out. More specifically, the following biometric measurements were performed: barrier function measuring the water evaporation density gradient of the skin with the Tewameter® TM 300 probe; sebum-regulating effect with Sebufix® F 16; and the effect on erythema by measuring the light reflected by the skin, using the Mexameter® MX 18 probe.

Lig-Syn-019 was formulated in a serum at 3% (w/w). The same formulation without the product was used as placebo.

A randomised and double-blind study was conducted with 20 volunteers in each experimental group, placebo and product. All the participants met the inclusion criteria defined: age above 18, oily skin with mild-moderate acne tendency, Caucasian ethnicity, adequate level of understanding of the clinical study, good state of health (physical and mental), having stopped using anti-acne or sebum-regulating products at least 7 days before the study in the experimental area.

The study duration was 28 days. Lig-Syn-019 and the placebo formulations were applied on the face in the morning and in the evening on a clean, dry skin, without rinsing and avoiding the eye contour. Biometric and dermatological evaluations were performed on day 0 and day 28 of the study.

As shown in Table 6, Lig-Syn-019 improved several biometric and dermatological parameters after 28 days of application compared to both placebo and day 0. More specifically, Lig-Syn-019 improved the barrier function, reduced the erythema, reduced the sebum, and reduced the presence of blackheads (small black/brown dots on the skin, with open clogged pores) and nodules (large (over 5 mm wide), painful lumps not filled with pus).

Table 6: Quantification of different skin parameters after 28 days of application of the product Lig-Syn-019 on the skin of volunteers with moderate-mild acne relative to placebo and to day 0.

These results indicate that the product improves the quality of acne-prone skin and prevents the formation of new breakouts and blemishes.

Example 9. Synthesis of ester derivates of Lignin

Several esters: Lignin linoleate, lignin stearate, and lignin ethylhexanoate were prepared with the following methodology.

Lignin (10 g) was first dissolved in THF (100 ml) and transferred to a round-bottom flask. Triethylamine (30 ml, 3.3 to 5 equiv. respect the hydroxyl groups) was added relatively fast. A solution of the corresponding acid chloride (1.1 to 1.3 equiv. respect the hydroxyl groups of the lignin) was dissolved in THF (50 ml) and added dropwise. The mixture was left stirring at room temperature for a minimum of two hours up to overnight. Once the reaction was finished, it was quenched with a cold aqueous solution of HCI (2-12%) by either adding it to the mixture or dropping the reaction into the acid. Two phases were formed and separated. The aqueous phase was washed three times with ethyl acetate or heptane. The organic fractions combined were washed with water and brine, dried over magnesium sulphate, filtered and concentrated under vacuum at 40 °C. The procedure yielded a mixture of esterified lignin and the corresponding free acid at an approximate 70:30 ratio. The ratio was calculated as follows: A known aliquot of the lignin was washed several times (3-9) with ethanol. The remaining solid and the ethanolic phase were dried separately and weighted. The ratio was calculated by dividing the weight of the ethanolic phase by the sum of weights.

Example 10. Preparation of mixtures of lignin esters and selected acids and characterisation

First, lignin esters were washed with EtOH several times until the product was pure (without free acids). Lignin esters purity was followed by H-NMR or HPLC. Then, the mixtures of Error! Reference source not found.7 were prepared by mixing the corresponding lignin ester pure with a 30% weight of the corresponding acid.

Lignin esters were used to prepare six different mixtures which are defined here:

Table 7: Mixtures prepared with the different lignin esters

Infrared Spectroscopy:

The characterization of lignin esters pure was carried out by Infrared Spectroscopy.

Although limited, this technique shows if the hydroxyl groups have been esterified by the disappearance of the broadband at 3500-3000 cm^-1.

The characteristic sharp bands between 2950 and 2800 cm^-1 correspond to the CH2 groups. FTIR main peak signals (cm-1):

Linoleate Lignin: 3008 - 2853, 1761 - 1739, 1653, 1592, 1508, 1462, 1266 - 1035, 913, 722.

Oleate Lignin: 3006 - 2852, 1761 - 1741 , 1592, 1508, 1462, 1360, 1198 - 1035, 966, 853, 722.

Stereate Lignin: 2919 - 2850, 1760 - 1741 , 1593, 1508, 1464, 1417, 1364, 1266 - 1035, 853, 720.

Ethylhexanoate Lignin: 2957 - 2860, 1755 - 1734, 1591, 1508, 1458, 1377, 1263 - 1033, 945, 851 , 729.

¹H Nuclear Magnetic Resonance spectroscopy

Instrument: Bruker BioSpin GmbH. Avance

Pulse sequence: zg30

Solvent: CDCI3

Temperature: 298 K

Scans: 32

Relaxation delay: 1 - 5 s

Pulse width: 8

Acquisition time: 2.6241

Width: 6250.0

Spectrometer Frequency: 400.20

FT size (Spectral Size): 65536

The Nuclear Magnetic Resonance (NMR) is used to see if the product is completely dry and to detect the presence of the corresponding free acid.

1 H Nuclear Magnetic Resonance spectroscopy of product 1: ¹H NMR (300 MHz, CDCh): 7.17 - 6.31, 5.56 - 5.04, 4.01 - 3.55, 2.83 - 2.72, 2.56, 2.35, 2.12 - 1.98, 1.74, 1.62, 1.43 - 1.20, 0.97 - 0.84.

1 H Nuclear Magnetic Resonance spectroscopy of product 2: ¹H NMR (300 MHz, CDCh): 7.18 - 6.52, 5.46 - 5.25, 3.96 - 3.65, 2.77, 2.55, 2.35, 2.05, 1.73, 1.62, 1.44 - 1.20, 0.97 - 0.82.

1 H Nuclear Magnetic Resonance spectroscopy of product 3: ¹H NMR (300 MHz, CDCI3): 7.21 - 6.49, 4.01 - 3.55, 2.56, 2.35, 1.74, 1.63, 1.25, 0.93 - 0.83.

1 H Nuclear Magnetic Resonance spectroscopy of product 4: ¹H NMR (300 MHz, CDCI3): 7.21 - 6.34, 4.00 - 3.60, 2.55, 2.35, 2.01 , 1.74, 1.62, 1.28, 0.97 - 0.83.

1 H Nuclear Magnetic Resonance spectroscopy of product 5: ¹H NMR (300 MHz, CDCI3): 7.12 - 6.32, 3.97 - 3.51, 2.55, 2.30, 1.36 - 1.12, 1.00 - 0.83.

1 H Nuclear Magnetic Resonance spectroscopy of product 6: ¹H NMR (300 MHz, CDCI3): 7.13 - 6.28, 4.01 - 3.42, 2.50, 2.29, 1.76 - 1.40, 1.31 , 1.02, 0.99 - 0.59. of mixtures of lignin esters and selected acids from

The antioxidant capacity of Oleic lignin and the 6 products of study was measured using the ABTS(2,2’-azinobis-(3-ethylbenzothiazoline-6-sulfonate) Assay. This assay was based on the interaction between antioxidants and the pre-formed green-blue stable radical cationic chromophore, ABTS*+. In the presence of antioxidants, the oxidized ABTS*+ radical was reduced to ABTS resulting in a discoloration of the solution, measured by the decrease in absorbance at 734 nm. Antioxidants scavenge ABTS*+ radical cation in a concentration dependent manner. In this assay, Trolox was used to standardize the sample TAG relative to Trolox (Trolox Equivalents Antioxidant Capacity, TEAC).

Briefly, 0.1 g of each sample was mixed with 1 mL of solvent (EtOH and DMSO) and left to dissolve overnight under shaking at RT in the dark. Then, the solution was centrifuged for 10 min at 10000 g and the supernatant was collected and subjected to ABTS analysis.

The results obtained for this study are shown in 9. Oleic lignin showed the highest antioxidant capacity, followed by product 1 , product 2, product 4, and product 3. Products 5 and 6 showed very low antioxidant activity.

Table 8. Total antioxidant capacity calculated for Oleic lignin and the 6 products of study EtOH and DMSO.

For reasons of completeness, various aspects of the invention are set out in the following numbered clauses:

1. A composition comprising a mixture of a modified lignin and oleic acid, wherein the modified lignin is a lignin wherein at least one of the hydroxyl groups is esterified with oleic acid; and wherein the modified lignin and the oleic acid are in a weight ratio from 80:20 to 60:40.

2. The composition according to clause 1 , wherein the lignin is Kraft lignin.

3. The composition according to clause 2, wherein the kraft lignin has a total hydroxyl content from 3 to 10 mmol% measured by ³¹PNMR.

4. The composition according to any of the clauses 1-3, wherein the degree of esterification of the hydroxyl groups of the modified lignin is equal to or higher than 90% measured by ³¹PNMR.

5. The composition according to any of the clauses 1-3, wherein all the hydroxyl groups of the modified lignin are esterified measured by ³¹PNMR.

6. The composition according to any of the clauses 1-5, wherein the modified lignin has a weight average molecular weight from 2,000 to 200,000 g/mol measured by size exclusion chromatography.

7. The composition according to any of the clauses 1-6, which is pharmaceutical or cosmetical composition, wherein the mixture of modified lignin and oleic acid are in an amount from 0.1 to 30 % by weight of the composition; the composition further comprises one or more pharmaceutically or cosmetically acceptable excipients or carriers, and, the sum of components, being 100% by weight.

8. The composition according to any of the clauses 1-7, wherein the modified lignin and the oleic acid are in a weight ratio from 95:5 to 40:60.

9. A composition as defined in any of the clauses 1-8, for use in the protection of a human, or animal living body, from ultraviolet radiation, wherein the composition is for topical application to the skin.

10. A composition as defined in any of the clauses 1-8, for use in the protection of a mammal, including a human, from oxidative stress caused by UV radiation, causing cell and tissue damage, wherein the composition is for topical application to the skin.

11. Use of a composition as defined in any of the clauses 1-8, for increasing the pigmentation in a mammal, including a human.

12. Use of a composition according to clauses 11 , wherein the increase in pigmentation is produced by increasing melanin production.

13. Use of the composition as defined in any of the clauses 1-8 to improve the preservation of cosmetic formulations.

14. Use of the composition as defined in any of the clauses 1-8, as film former.

15. A composition as defined in any of the clauses 1-8, for use in the prevention and/or treatment of acne.

16. A process for preparing the composition as defined in any of the clauses 1-8, which comprises reacting lignin with an excess of a compound selected from oleyl halide and oleyl anhydride with respect to the hydroxyl groups of the lignin, in an appropriate solvent. Citation List

Patent Literature

• W02015/094099A1

Non-Patent Literature

• New products from lignin by Oihana Gordobil Goni, published in San Sebastian in 2018.

• Lewis, H., Brauns, F., Buchanan, M., and Brookbank, E. (1943). "Lignin esters of mono-and dibasic aliphatic acids," Industrial & Engineering Chemistry 35(10),

1113-1117.

• Pawar, S. N., Venditti, R. A., Jameel, H., Chang, H. M., and Ayoub, A. (2016). "Engineering physical and chemical properties of softwood kraft lignin by fatty acid substitution," Industrial Crops and Products 89, 128-134. • E. L. Hult, J. Ropponen, K. Poppius-Levlin, T. Ohra-Aho and T. Tamminen,

Enhancing the barrier properties of paper board by a novel lignin coating, Ind. Crops Prod., 2013, 50, 694-700.

• Xianzhi Meng et al., “Determination of hydroxyl groups in biorefinery resources via quantitative ³¹ P NMR spectroscopy”, Nature Protocols, vol. 14, pp. 2627-2647.

Claims

Claims

1. A composition comprising a mixture of a modified lignin and a (Cs-Cis) carboxylic acid, wherein the modified lignin is a lignin wherein at least one of the hydroxyl groups is esterified with an acid selected from the group consisting of oleic acid, linoleic acid, stearic acid, 2- ethylhexanoic acid; the (C₈-Ci₈) carboxylic acid is selected from the group consisting of oleic acid, linoleic acid, stearic acid, 2-ethylhexanoic acid; and the modified lignin and the (Cs-Cis) carboxylic acid are in a weight ratio from 80:20 to 60:40.

2. The composition according to claim 1, comprising a mixture of a modified lignin and oleic acid, wherein the modified lignin is a lignin wherein at least one of the hydroxyl groups is esterified with oleic acid; and wherein the modified lignin and the oleic acid are in a weight ratio from 80:20 to 60:40.

3. The composition according to any of the claims 1-2, wherein the lignin is Kraft lignin.

4. The composition according to claim 3, wherein the kraft lignin has a total hydroxyl content from 3 to 10 mmol% measured by ³¹PNMR.

5. The composition according to any of the claims 1-4, wherein the degree of esterification of the hydroxyl groups of the modified lignin is equal to or higher than 90% measured by ³¹PNMR.

6. The composition according to any of the claims 1-5, wherein all the hydroxyl groups of the modified lignin are esterified measured by ³¹PNMR.

7. The composition according to any of the claims 1-6, wherein the modified lignin has a weight average molecular weight from 2,000 to 200,000 g/mol measured by size exclusion chromatography.

8. The composition according to any of the claims 1-7, which is pharmaceutical or cosmetical composition, wherein the mixture of modified lignin and oleic acid are in an amount from 0.1 to 30 % by weight of the composition; the composition further comprises one or more pharmaceutically or cosmetically acceptable excipients or carriers, and, the sum of components, being 100% by weight.

9. The composition according to any of the claims 1-8, wherein the modified lignin and the oleic acid are in a weight ratio from 95:5 to 40:60.

10. The composition according to claim 1, wherein the mixture of a modified lignin and a (C₈-Ci₈) carboxylic acid is selected from the following ones: a) linoleate lignin and linoleic acid; b) oleate lignin and Linoleic acid; c) stereate lignin and stearic acid; d) stereate lignin and oleic acid; e) stereate lignin and 2-ethylhexanoic acid; and f) ethlyhexanoate lignin and 2-ethylhexanoic acid.

11. A composition as defined in any of the claims 2-9, for use in the protection of a human, or animal living body, from ultraviolet radiation, wherein the composition is for topical application to the skin.

12. A composition as defined in any of the claims 2-9, for use in the protection of a mammal, including a human, from oxidative stress caused by UV radiation, causing cell and tissue damage, wherein the composition is for topical application to the skin.

13. Use of a composition as defined in any of the claims 2-9, for increasing the pigmentation in a mammal, including a human.

14. Use of a composition according to claim 13, wherein the increase in pigmentation is produced by increasing melanin production.

15. Use of the composition as defined in any of the claims 2-9 to improve the preservation of cosmetic formulations.

16. Use of the composition as defined in any of the claims 2-9, as film former.

17. Use of the composition as defined in any of the claims 1-10, as an antioxidant.

18. A composition as defined in any of the claims 2-9, for use in the prevention and/or treatment of acne.

19. A process for preparing the composition as defined in any of the claims 2-9, which comprises reacting lignin with an excess of a compound selected from oleyl halide and oleyl anhydride with respect to the hydroxyl groups of the lignin, in an appropriate solvent.