FIELD OF THE INVENTION

The present invention describes compounds of formula (I) of the betaxanthin family, specifically betaxanthins derived from tryptophan and phenylethylamine, or pharmaceutical compositions comprising the same, for use in the treatment and/or prevention of cancer and/or tumours. In addition, the use of nutraceutical and food compositions for the prevention of cancer or as chemopreventive compositions, comprising said compounds of formula (I), is described.

BACKGROUND OF THE INVENTION

Cancer is a common and occasionally lethal disease. As human populations age, the incidence of cancer increases, and with it the urgency to develop more potent and less toxic antineoplastic drugs. Plants are a source of phytochemicals which can be molecules with improved safety and effectiveness in chemoprevention and treatments for improving the health of oncological patients. Certain phytochemicals can induce biological responses in animal cells, including inhibition of enzymes, antioxidant activity and regulation of cell signalling pathways (Pratheeshkumar et al., BioMed Research International, 2015. DOI: 10.1155/2015/324021).

Betalains are water-soluble nitrogenated vegetable pigments which are characteristic of plants in the order Caryophyllales. This type of compounds are produced when betalamic acid -the structural unit of all betalains- is fused with amines or amino acids (Gandia-Herrero & Garcia-Carmona, Trends in Plant Science, 2013. DOI: 10.1016/j.tplants.2013.01.003).

Generally, betalains are classified into two groups according to their colour. Betacyanins have a violet hue, while betaxanthins have a yellow hue. However, their colour indicates little or nothing about the chemical structure of the molecule and its functional groups, being said structure and groups responsible for their chemical capacity and biological activity.

From a structural standpoint betalains may be classified into two large groups: (1) those with a net positive charge which comprise in their structure a quaternary ammonium group (iminium), such as betacyanins or indicaxanthin (see Figure 1); and (2) those comprising in their structure an imine group without a net positive charge, among which are vulgaxanthins (I, II, III, IV), tryptophan-betaxanthin, phenylethylamine-betaxanthin, phenylalanine- betaxanthin, and dopaxanthin (represented in Figure 2). Among betaxanthins, a small group having a primary amine bonded by an alkyl chain to an aromatic ring, may be recognized, such as tryptophan-betaxanthin, phenylethylamine-betaxanthin, phenylalanine-betaxanthin or dopaxanthin, which are structurally very different from the betaxanthins having a linear amine, such as vulgaxanthins (l-IV) or humilixanthin (represented in Figure 2). These betaxanthins having an aromatic ring represent 7 molecules of the total of 31 identified in nature (cf. Table 1 of Gandia-Herrero & Garcia-Carmona, Trends in Plant Science, 2013. DOI: 10.1016/j.tplants.2013.01.003). In addition, the betacyanines represented in Figure 1 comprise groups R _a and R _b , which can be a complex sugar and/or acid group structure. These various structural characteristics mean that their behaviour may be markedly different, even if they are classified within the same colour group.

The prior art describes different in vitro studies that show the antioxidant activity of extracts containing betalains. Antioxidants act as electron donors that neutralize reactive oxygen species and other free radicals that would otherwise potentially damage DNA and lead to oncogenesis. Numerous studies on both cells and in preclinical trials support the idea that antioxidant molecules can protect cells against carcinogenesis (Borek, Integrative cancer therapies, 2004. DOI: 10.1 177/1534735404270578; ES2349522, 2011 , Garcia-Carmona et ai.\ Gandia-Herrero & Garcia-Carmona, Trends in plant science, 2013. DOI: 10.1016/j.tplants.2013.01.003). However, other studies link the proliferation of certain types of cancers to antioxidants, since factor p53 causes an increase in the reactive oxygen species, triggering apoptosis; and consequently, the disappearance of these reactive species due to the antioxidants prevents apoptosis and leads to cell proliferation (Valko et al., Chemico-biological interactions, 2006. DOI 10.1016/j.cbi.2005.12.009; Sayin et al., Science translational medicine, 2014. DOI: 10.1126/scitranslmed.3007653; Perera & Bardeesy, Nature, 201 1. DOI: 10.1038/475043a). These mixed results indicate that specific studies for each molecule classified as an antioxidant are required, since it is not possible to generalize that a molecule with, or being structurally similar to, another molecule having in vitro antioxidant effects, will be effective as an anti-tumour agent. The relationship between antioxidant capacity and anti-tumour activity is not direct. Anti-tumour activity is more complex and can obey to different biochemical and structural factors for each tested molecule, which may not be linked to its antioxidative power.

On the other hand, the prior art describes plant extracts containing unpurified betalains, mainly betacyanins, such as betanine or betanidine (ES2320380; US2003036565; US6312697B1) for the treatment of cancer, due to the fact that some betacyanins have shown antioxidating, antiinflammatory, hepatoprotective, anticancerous, antidiabetic, antilipidemic, antimicrobian, radio-protective, and anti-proliferative properties (Gandia- Herrero, Escribano & Garcia-Carmona, Critical Reviews in Food Science, 2016. DOI: 10.1080/10408398.2012.740103). In particular, several publications show the anticarcinogenic and chemopreventive activity of various extracts containing betalains (Kapadia et al., Cancer leters, 1996. DOI: 10.1016/0304-3835(65)04087-0; Kapadia et al., Anti-Cancer Agents in Medicinal Chemistry, 2011. DOI: 10.2174/187152011795347504; SreeKanth et al., International Journal of Phytochemistry, 2007. DOI: 10.1016/j.phymed.2007.03.017; Kapadia et al., Pharmacological Research, 2003. DOI: 10.1016/S1043-6618(02)00285-2, Lechner et al., Journal of Medical Food, 2010. DOI: 10.1089/jmf.2008.0280 y Zou et ai, Nutrition Journal, 2005. DOI: 10.1186/1475-2891-4-25). Moreover, extracts of Opuntia fruits (whose main pigment is indicaxanthin) have also shown potential in the protection and recovery of the liver after inducing damage in said organ (Galati et ai., Phytotherapy Research, 2005. DOI: 10.1002/ptr.1741 ) . In general, said vegetable extracts contain mainly betacyanins, and the nature of the bioactive molecules present in said extracts has not been appropiately explained.

However, some studies on cancerous cell lines, that use partially purified betalains from plant extracts, have been described. On one hand, Farabegoli etai., (Farabegoli et ai., Food chemistry, 2017. DOI: 10.1016/j.foodchem.2016.09.1 12) partially purified a beetroot extract obtaining two fractions rich in betalains, named fractions R1 and R2. In fraction R1 Farabegoli et ai. identified vulgaxanthin I (glutamine-betaxanthin) and in fraction R2 betanin, isobetanin and betanidin. No other betalain was mentioned by the authors. Moreover, Farabegoli et al. do not show antioxidant or anticarcinogenic activity with individual compounds, but instead with the group of betalains and other molecules present, respectively, in fractions R1 and R2. Similarly, in the article by Khan et al. (Khan et ai, LWT- Food Science and Technology, 2012. DOI: 10.1016/j.lwt.2012.01.025) two betalain-rich fractions are obtained from berries of Rivina humilis, among which were identified: prolin- betaxanthin (indicaxanthin), 3,4-dihydroxyphenylalanine-betaxanthin (dopaxanthin), glutamic acid-betaxanthin (vulgaxanthin II), glutamine-betaxanthin (vulgaxanthin I), aspartic acid-betaxanthin, 5-hydroxynorvaline-betaxanthin (humilixanthin), betanin, betanidin, thyrosin-betaxanthin and dopamin-betaxanthin (c.f. Khan et al., Table 1 , page 318). These authors describe the cytotoxic action of the extract of Rivina humilis berries on cancerous cell lines, but not that of the individual components, being said individual components pigments ornot.

Tryptophan-betaxanthin (Figure 2) is a betaxanthin without net positive charge and with an aromatic ring, where the betalamic acid is condensed with the aminoacid tryptophan (Gandia-Herrero & Garcia-Carmona, Trends in Plant Science, 2013. DOI: 10.1016/j.tplants.2013.01.003). This compound is present at a trace level in the plant Celosia argentea (Schliemann et al., Phytochemistry, 2001. DOI: 10.1016/S0031- 9422(01)00141-8) and is found in plants of traditional Chinese medicine (TCM)).

Other betaxanthins, such as phenylethylamine-betaxanthin and phenylalanine-betaxanthin (Figure 2), are present in very small amounts in some varieties of the fruits of Opuntia ficus- indica, even though indicaxanthin (Figure 1) is the main pigment (Gandia-Herrero & Garcia- Carmona, Trends in Plant Science, 2013, DOI: 10.1016/j.tplants.2013.01.003). On the other hand an in vitro antioxidant effect has been described for tryptophan-betaxanthin as a pure molecule, from which no bioactive or anti-tumour in vivo effect can be inferred (Cai, Sun & Corke, Journal of Agricultural and Food Chemistry, 2003. DOI: 10.1021/jf030045u). However, the same authors describe the difficulty of obtaining this pure pigment (cf. Cai, Sun & Corke, Tabla 1).

On the other hand, in silico studies describe tryptophan-betaxanthin as a potential inhibitor of the protein Sirtl (silent information regulator 1) (Chen et ai, Journal of Biomolecular Structure and Dynamics, 2012. DOI: 10.1080/07391102.2012.726191). Silent information regulator 1 (Sirtl) is a class III nicotinamide dinucleotide adenine dependent deacetylase histone. The information supplied was limited to computational molecular modelling studies, with no experimental measurements or access to the pure molecule and without giving biological evidence of any potentially positive activity.

Finally, a study performed by “in silico” molecular dynamic simulation, suggests that tryptophan-betaxanthin may be a PPAR agonist (Chen et ai, Journal of Biomolecular Structure and Dynamics, 2012. DOI: 10.1080/07391102.2012.726191), since said molecule obtained the highest coupling score, in silico, compared to the other molecules tested computationally, but without experimental evidence of any PPAR mediated effect.

BRIEF DESCRIPTION OF THE INVENTION

An aspect of the present invention refers to a compound of formula (I), or an enantiomer, diastereomer or E/Z isomer thereof:

wherein,

Ri is H, -COOH, or -COOR ₆ , wherein R ₆ is a C ₁ -C ₆ alkyl group;

R ₂ is a C ₁ -C ₁₀ unsubstituted alkanediyl group or a C ₁ -C ₁₀ alkanediyl group having one or more substituents independently selected from the group consisting of: halogen, Ci- C6 alkyl, -R ₄ -COR ₅ and -R ₄ -COOR ₅ , wherein R ₄ is a C ₁ -C ₆ alkanediyl group and R ₅ is - H or a C ₁ -C ₆ alkyl group;

R ₃ is selected from the group consisting of:

an unsubstituted phenyl group or a phenyl group having one OH substituent or having one or more substituents independently selected from the group consisting of: halogen, C ₁ -C ₆ alkyl, -OR ₆ , -COOH and -COOR ₆ , wherein R ₆ is a C ₁ -C ₆ alkyl group;

or

an unsubstituted 1 H-indol-3-yl group or a 1 H-indol-3-yl group having one or more substituents independently selected from the group consisting of: OH, halogen, C ₁ -C ₆ alkyl, -OR ₆ , -COOH and -COOR ₆ , wherein R ₆ is an C ₁ -C ₆ alkyl group;

and wherein when R ₃ is an unsubstituted phenyl group, Ri is H;

for use in the treatment and/or prevention of cancer and tumours in humans and animals.

Optionally, said compound of formula (I) for use, is administered in combination with other therapeutic and/or preventive treatments.

Another aspect of the invention refers to a compound of formula (I), or an enantiomer, diastereomer or E/Z isomer thereof:

wherein,

- Ri is H or -COOH;

R ₂ is an unsubstituted C ₁ -C ₁₀ alkanediyl group;

R ₃ is selected from:

an unsubstituted phenyl group; or

an unsubstituted 1 H-indol-3-yl group;

and wherein when R3 is an unsubstituted phenyl group, Ri is H;

for use in the treatment and/or prevention of cancer and tumours in humans and animals.

Optionally, said compound of formula (I) for use, is administered in combination with other therapeutic and/or preventive treatments.

Yet another aspect of the present invention refers to a pharmaceutical composition comprising the compound of formula (I), or an enantiomer, diastereomer or E/Z isomer thereof, described in the present invention, and at least one pharmaceutically acceptable excipient, for use in the treatment and/or prevention of cancer and tumours in humans and animals. Optionally, said pharmaceutical composition for use, is administered optionally in combination with other therapeutic and/or preventive treatments.

Finally, another aspect of the present invention refers to the use of a nutraceutical or food composition comprising a compound of formula (I), or an enantiomer, diastereomer or E/Z isomer thereof, as described in the present invention, and at least one acceptable food excipient, for the prevention of cancer or as a chemopreventive agent.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 : Chemical structure of betalains with net positive charge.

Figure 2: Chemical structure of betalains with no net positive charge having an amine linked to an aromatic ring, such as tryptophan-betaxanthin, phenylethylamine-betaxanthin, phenylalanine-betaxanthin or dopaxanthin and those derived from a linear amine, such as vulgaxanthin I or humilixanthin.

Figure 3: In vitro antioxidant effect of the compounds described in the present description compared to that of betalamic acid.

Figure 4: In vivo antioxidant effect of the compounds described in the present description. The assay was performed by measuring the capacity of each compound to reduce fluorescence in the pharynx of strainTJ375 of the nematode C. elegans, as a measure of oxidative stress, when exposed to juglone. (A) Histogram of the in vivo antioxidant activity, (B) representative image representing of control exposed to juglone, (C) representative image of an animal treated with 25 mM dopaxanthin and exposed to juglone.

Figure 5: Method for measuring tumour size in vivo : (A) C. elegans (wild-type, N2), with normal gonad. (B) C. elegans (JK1466 mutant) with tumour in gonad. The gonads of both specimens were marked from the loop region to the proximal region. Scale bar 100 pm. This measurement was used for the evaluation of tumour reduction with bioactive compounds.

Figure 6: Histogram of gonads surface areas of animals with wild type phenotype (N2), untreated tumoral phenotype (JK1466) and of those treated with tryptophan-betaxanthin, phenylethylamine-betaxanthin, phenylalanine-betaxanthin and dopaxanthin. ^Statistically significant changes.

Figure 7: Histograms and survival curves for C. elegans JK1466 control (untreated) and treated with tryptophan-betaxanthin (A and B) and phenylethylamine-betaxanthin (C and D). ^Statistically significant changes.

DESCRIPTION

The present invention refers to a compound of formula (I), or an enantiomer, diastereomer or E/Z isomer thereof:

wherein,

Ri is H, -COOH, or -COOR ₆ , wherein R ₆ is a C1-C6 alkyl group;

R ₂ is a C ₁ -C ₁₀ unsubstituted alkanediyl group or a C ₁ -C ₁₀ alkanediyl group having one or more substituents independently selected from the group consisting of: halogen, Ci- C6 alkyl, -R ₄ -COR ₅ and -R ₄ -COOR ₅ , wherein R ₄ is a C ₁ -C ₆ alkanediyl group and R ₅ is - H or a C1-C6 alkyl group;

R3 is selected from:

an unsubstituted phenyl group or a phenyl group having one OH substituent or having one or more substituents independently selected from the group consisting of: halogen, C1-C6 alkyl, -OR ₆ , -COOH and -COOR ₆ , where R ₆ is a C1-C6 alkyl group;

or

an unsubstituted 1 H-indol-3-yl group or a 1 H-indol-3-yl group having one or more substituents independently selected from the group consisting of: OH, halogen, C1-C6 alkyl, -OR6, -COOH and -COOR6, wherein R6 is an C ₁ -C ₆ alkyl group; and wherein when R ₃ is an unsubstituted phenyl group, Ri is H;

for use in the treatment and/or prevention of cancer and tumours in humans and animals,

Optionally, said compound of formula (I) for use, is administered in combination with other therapeutic and/or preventive treatments.

The term“comprises” indicates that includes a group of certain features (for example a group of features A, B and C) is interpreted as meaning that it includes those features (A, B and C), but that it does not exclude the presence of other features (for example features D or E), as long as they do not render the claim unworkable. Additionally, the terms “contains”,“includes”,“has” or“encompass”, and the plural forms thereof, should be taken as synonymous of the term“comprises” for the purposes of present invention. On the other hand, if the wording "consist of" is used, then no further features are present in the apparatus/method/product apart from the ones following said wording. In this sense, for the purposes of present invention, the term“comprises” may be replaced by any of the terms “consist of”, or“consists essentially of”. Accordingly,“comprises” may refer to a group of features A, B and C, which additionally may include other features, such as E and D, with the condition that said features do not render the claim unworkable, but said term “comprises” also includes the situation in which the group of features “consists of” or “consists essentially” of A, B and C.

The compounds of formula (I) disclosed in present description also include any pharmaceutical salt or solvate thereof.

The term "pharmaceutically acceptable salt" refers to any pharmaceutically acceptable salt, which upon administration to the patient is capable of providing (directly or indirectly) a compound as described herein. Such salts preferably are acid addition salts with physiologically acceptable organic or inorganic acids, or alkali addition salts with physiologically acceptable organic or inorganic bases. Examples of the acid addition salts include mineral acid addition salts such as, for example, hydrochloride, hydrobromide, hydroiodide, sulphate, nitrate, phosphate, and organic acid addition salts such as, for example, acetate, trifluoroacetate, maleate, fumarate, citrate, oxalate, succinate, tartrate, malate, mandelate, methanesulphonate and p-toluenesulphonate. Examples of the alkali addition salts include inorganic salts such as, for example, sodium, potassium, calcium and ammonium salts, and organic alkali salts such as, for example, ethylenediamine, ethanolamine, N,N-dialkylenethanolamine, triethanolamine and basic aminoacids salts. However, it will be appreciated that non-pharmaceutically acceptable salts also fall within the scope of the invention since those may be useful in the preparation of pharmaceutically acceptable salts. Procedures for salt formation are conventional in the art.

The term "solvate" in accordance with this invention should be understood as meaning any form of the active compound in accordance with the invention in which said compound is bonded by a non-covalent bond to another molecule (normally a polar solvent), including especially hydrates and alcoholates.

Preferably, R ₂ is an unsubstituted C ₁ -C ₁₀ alkanediyl group. More preferably, R ₂ is -CH ₂ -. The term“alkanediyl” refers to a alkane saturated divalent group -(Chh for the purposes of present invention. Non-limiting examples of alkanediyl groups are methylene -CH ₂ -, ethylene -CH2-CH2-, propylene -CH2-CH2-CH2-, etc.

Also, preferably, R ₃ is selected from:

an unsubstituted phenyl group or a phenyl group having one or more substituents independently selected from the group consisting of: halogen, C ₁ -C ₆ alkyl, -OR ₆ , - COOH and -COOR ₆ , wherein R ₆ is a C ₁ -C ₆ alkyl group;

or

an unsubstituted 1 H-indol-3-yl group or a 1 H-indol-3-yl group having one or more substituents selected independently from the group consisting of: OH, halogen, C ₁ -C ₆ alkyl, -OR ₆ , -COOH and -COOR ₆ , wherein R ₆ is an C ₁ -C ₆ alkyl group;

Also, preferably, R ₃ is selected from:

an unsubstituted phenyl group;

or

an unsubstituted 1 H-indol-3-yl group or a 1 H-indol-3-yl group having one or more substituents independently selected from the group consisting of: OH, halogen, C ₁ -C ₆ alkyl, -OR ₆ , -COOH and -COOR ₆ , wherein R ₆ is an C ₁ -C ₆ alkyl group;

Also, preferably, R ₃ is selected from:

an unsubstituted phenyl group;

or

an unsubstituted 1 H-indol-3-yl group or a 1 H-indol-3-yl group having one or more substituents independently selected from the group consisting of: halogen, C ₁ -C ₆ alkyl, -ORe, -COOH and -COOR ₆ , wherein R ₆ is a C ₁ -C ₆ alkyl group;

Also, preferably, R ₃ is selected from:

an unsubstituted phenyl group or a phenyl group having one or more substituents independently selected from the group consisting of: halogen, C ₁ -C ₆ alkyl, -OR ₆ , - COOH and -COOR ₆ , wherein R ₆ is a C ₁ -C ₆ alkyl group;

or an unsubstituted 1 H-indol-3-yl group.

More preferably, the C ₁ -C ₆ alkyl group is methyl or ethyl.

Also, more preferably, R ₃ is selected from:

an unsubstituted phenyl group;

or

an unsubstituted 1 H-indol-3-yl group.

Thus, the present invention also refers to the use of the enantiomers of the compound of formula (I), having the opposite stereochemical configurations with respect to the two chiral centers marked with an asterisk (*) in present application.

In addition, the present invention also refers to the use of the diastereomers of the compound of formula (I), which have opposite stereochemical configurations with respect to only one the two chiral centers marked with an asterisk (*) in present application.

Finally, the present invention also refers to the use of all or any of the E/Z isomers of the compound of formula (I) with regard to the configuration of the substituents of the carbon- carbon double bond.

Due to their activity, the compounds of formula (I) can be used for the treatment and/or prevention of cancer in pharmaceutical compositions, and also as chemopreventive agents and in the prevention of cancer, in nutraceutical and/or food compositions.

The compounds of formula (I) feature antioxidant activity as well as anti-tumour activity.

Specifically, the present invention shows the in vivo anti-tumour effects of the compounds of formula (I), exemplified by tryptophan-betaxanthin and phenylethylamine-betaxanthin, in the animal model Caenorhabditis elegans (nematode) (Figure 6). It should be noted that in- depth studies, such as those presented in the invention, have not until now been possible, since the levels of tryptophan-betaxanthin in plants are very low and not viable for an in vivo assay. The compounds of present invention were synthesized using the biotechnological method described by Guerrero-Rubio et al., (Guerrero-Rubio etai, Microbial Biotechnology, 2019. DOI: 10.11 11/1751-7915.13452). In this way it was possible to evaluate individually each of the betaxanthins of the invention, conducting an in-depth study of its anti-tumour effects on an animal model.

More specifically, this invention describes the anti-tumour and antiproliferative effect of the compounds of formula (I) using the animal model C. elegans, specifically the JK1466 mutant strain (Figures 5 and 6). This strain of C. elegans has a mutation of the tumour suppression gene gld-1. In gld-1 (q485) mutants the germ cells cannot leave the mitosis phase and continue proliferating throughout the gonad, forming a germ line tumour that is lethal to the animal (Francis et al., Genetics, 1995, 139(2): 579-606). An objective evaluation method has been developed for tumour growth in this strain to determine the potential anti-tumour activity of the molecules of the invention, in vivo.

In addition, to confirm the existence of a direct relationship between the antioxidant effect of the compounds of formula (I) of the invention and their anti-tumour effects, the antioxidant capacity thereof has been assayed both in vitro and in vivo (Figures 3 and 4).

The in vitro assay was performed by ABTS assay (Re et al., Free radical biology and medicine, 1999. DOI: 10.1016/S0891 -5849(98)00315-3). The in vivo assay was performed with the TJ375 mutant strain of C. elegans. This strain has the GFP protein fused to the heat shock proteins expressed in the pharynx of C. elegans (Guerrero-Rubio et al., Food Chemistry, 2019. DOI: 10.1016/j.foodchem.2018.09.067). The animals were treated with the test compound and then exposed to oxidative stress caused by juglone (naftoquinone). The lower the activation of the heat shock proteins is, the greater the antioxidant capacity of the compound is. The oxidative stress state of the animal is evaluated by the area and intensity of fluorescence associated with the accumulation of fluorescent HSP.

Betalains with an imine structure, without charge, which contain an aromatic ring and obtained from the corresponding primary amine, have been used such as: tryptophan- betaxanthin, phenylethylamine-betaxanthin, phenylalanine-betaxanthin and dopaxanthin, synthesized by a biotechnological process. The biotechnological production was performed in microbial factories following a method previously described by Guerrero-Rubio et al., Microbial Biotechnology, 2019 (DOI: 10.1 111/1751-7915.13452).

Although the molecules belonging to the betaxanthin family have a similar structure, small structural differences were crucial for obtaining antioxidant and also anti-tumour activity.

Therefore, this invention shows experimentally that the compounds of formula (I), exemplified by tryptophan-betaxanthin and phenylethylamine-betaxanthin, have an therapeutic effect promoting health and more specifically an anti-tumour effect, wherein said therapeutic effect cannot be directly connected with their antioxidant capacity.

Specifically, dopaxanthin showed the greatest capacity to reduce oxidative stress in vivo, yet showed no anti-tumour capacity. However, tryptophan-betaxanthin was the molecule with the lowest capacity to reduce oxidative stress in vivo, but showed the highest efficacy to reduce tumour size in C. elegans and is a candidate for new treatment and/or prevention strategies in diseases such as cancer.

The prior art (ES2349522B2, or Khan et al., FTW -Food Science and Technology, 2012. DOI: 10.1016/j.lwt.2012.01.025) suggests that the antioxidant capacity of betalains is linked to the presence of H donor groups, such as the imine groups present in all of them, or to a greater number of hydroxyl groups (cf. ES2349522B2 page 3 lines 2-6 and Khan et al., page 320 column 2, last lines). However, it appears that the prior art is contradictory, as patent ES2349522 B2 indicates that betaxanthins exhibit greater antioxidant capacity than betacyanins (cf. ES2349522 B2, page 3 lines 2-3), while Farabegoli et al. describe the opposite (cf. Farabegoli et ai, page 359, column 2, last paragraph of section 3.1).

In addition, as previously indicated and as shown in present application, even though a molecule may show, or potentially show, antioxidant activity in vitro, it is not possible to generalize that said molecule will be effective accordingly as an anti-tumour agent.

The presence of hydroxyls, as suggested by both Khan et al., such as patent ES2349522 B2, does not seem to help select betalains with anticarcinogenic activity, as the compound phenylethylamine-betaxanthin, for use in the treatment and/or prevention of cancer according to the present invention, shows anti-tumour activity, while dopaxanthin, which has a greater number of hydroxyls, does not show such activity.

A preferred embodiment of the invention refers to a compound of formula (I) for use, wherein:

- Ri is -COOH;

R2 is -CH2-; and

R3 is selected from:

a phenyl group having one OH substituent or having one or more substituents independently selected from the group consisting of: halogen, C1-C6 alkyl, -OR ₆ , - COOH and -COOR ₆ , wherein R ₆ is a C1-C6 alkyl group;

or

an unsubstituted 1 H-indol-3-yl group or a 1 H-indol-3-yl group having one or more substituents independently selected from the group consisting of: OH, halogen, C1-C6 alkyl, -OR ₆ , -COOH and -COOR ₆ , wherein R ₆ is an C1-C6 alkyl group;

and the compound has formula (II):

Preferably, R ₃ is selected from:

a phenyl group having one or more substituents independently selected from the group consisting of: halogen, C ₁ -C ₆ alkyl, -OR ₆ , -COOH and -COOR ₆ , wherein R ₆ is a C ₁ -C ₆ alkyl group;

or

an unsubstituted 1 H-indol-3-yl group or a 1 H-indol-3-yl group having one or more substituents independently selected from the group consisting of: OH, halogen, C ₁ -C ₆ alkyl, -OR ₆ , -COOH and -COOR ₆ , wherein R ₆ is an C ₁ -C ₆ alkyl group;

Also, preferably, R ₃ is selected from:

a phenyl group having one or more substituents independently selected from the group consisting of: halogen, C ₁ -C ₆ alkyl, -OR ₆ , -COOH and -COOR ₆ , wherein R ₆ is a C ₁ -C ₆ alkyl group;

or

an unsubstituted 1 H-indol-3-yl group or a 1 H-indol-3-yl group having one or more substituents independently selected from the group consisting of: halogen, C ₁ -C ₆ alkyl, -ORe, -COOH and -COOR ₆ , wherein R ₆ is a C ₁ -C ₆ alkyl group;

Also, preferably, R ₃ is selected from:

a phenyl group having one or more substituents independently selected from the group consisting of: halogen, C ₁ -C ₆ alkyl, -OR ₆ , -COOH and -COOR ₆ , wherein R ₆ is a C ₁ -C ₆ alkyl group;

or

an unsubstituted 1 H-indol-3-yl group.

A more preferred embodiment refers to a compound of formula (II) for use according to the present invention, wherein R ₃ is an unsubstituted 1 H-indol-3-yl group and the compound of formula (II) is tryptophan-betaxanthin:

tryptophan-betaxanthin Another preferred embodiment refers to a compound of formula (I) for use according to the present invention, wherein:

- Ri is H;

R2 is -CH2-; and

R ₃ is selected from:

an unsubstituted phenyl group or a phenyl group having one OH substituent or having one or more substituents independently selected from the group consisting of: halogen, C ₁ -C ₆ alkyl, -OR ₆ , -COOH and -COOR ₆ , wherein R ₆ is a C ₁ -C ₆ alkyl group;

or

an unsubstituted 1 H-indol-3-yl group or a 1 H-indol-3-yl group having one or more substituents independently selected from the group consisting of: OH, halogen, C ₁ -C ₆ alkyl, -OR ₆ , -COOH and -COOR ₆ , wherein R ₆ is an C ₁ -C ₆ alkyl group;

and the compound has formula (III):

Preferably R ₃ is selected from:

an unsubstituted phenyl group;

or

an unsubstituted 1 H-indol-3-yl group or a 1 H-indol-3-yl group having one or more substituents independently selected from the group consisting of: OH, halogen, C ₁ -C ₆ alkyl, -OR ₆ , -COOH and -COOR ₆ , wherein R ₆ is an C ₁ -C ₆ alkyl group;

Also, preferably, R ₃ is selected from:

an unsubstituted phenyl group;

or

an unsubstituted 1 H-indol-3-yl group or a 1 H-indol-3-yl group having one or more substituents independently selected from the group consisting of: halogen, C ₁ -C ₆ alkyl, -ORe, -COOH and -COOR ₆ , wherein R ₆ is a C ₁ -C ₆ alkyl group; Another more preferred embodiment refers to a compound of formula (III) for use according to the present invention, wherein R3 is an unsubstituted phenyl group, and the compound of formula (III) is phenylethylamine-betaxanthin:

Another aspect of the invention refers to a pharmaceutical composition comprising compound of formula (I), or an enantiomer, diastereomer or E/Z isomer thereof:

wherein,

Ri is H, -COOH, or -COOR ₆ , wherein R ₆ is a C1-C6 alkyl group;

R ₂ is a C ₁ -C ₁₀ unsubstituted alkanediyl group or a C ₁ -C ₁₀ alkanediyl group having one or more substituents independently selected from the group consisting of: halogen, Ci- C6 alkyl, -R ₄ -COR ₅ and -R ₄ -COOR ₅ , wherein R ₄ is a C ₁ -C ₆ alkanediyl group and R ₅ is - H or a C1-C6 alkyl group;

R3 is selected from:

an unsubstituted phenyl group or a phenyl group having one OH substituent or having one or more substituents independently selected from the group consisting of: halogen, C1-C6 alkyl, -OR ₆ , -COOH and -COOR ₆ , wherein R ₆ is a C1-C6 alkyl group;

or an unsubstituted 1 H-indol-3-yl group or a 1 H-indol-3-yl group having one or more substituents independently selected from the group consisting of: OH, halogen, C1-C6 alkyl, -OR ₆ , -COOH and -COOR ₆ , wherein R ₆ is an C1-C6 alkyl group;

and wherein when R3 is an unsubstituted phenyl group, Ri is H;

and at least one pharmaceutically acceptable excipient, for use in the treatment and/or prevention of cancer and tumours in humans and in animals.

Thus, the present invention refers to the use of a compound of formula (I) or of an enantiomer, diastereomer or E/Z isomer thereof, more preferably the use of a compound of formula (II) or the use of a compound of formula (III), even more preferably the use of tryptophan-betaxanthin or phenylethylamine-betaxanthin, or of a pharmaceutical composition comprising the same, in the manufacturing of a medicament for the treatment and/or prevention of cancer and tumours in humans and in animals, optionally in combination with other therapeutic and/or preventive treatments.

Additionally, present invention refers to a method for treatment and/or prevention of cancer and tumours in humans and animals, which comprises the administration of a pharmaceutically effective amount of a compound of formula (I), or of an enantiomer, diastereomer or E/Z isomer thereof, more preferably of a compound of formula (II) or of a compound of formula (III), even more preferably of tryptophan-betaxanthin or phenylethylamine-betaxanthin, to a subject in need thereof, or of a pharmaceutical composition comprising the same. Optionally, said method further comprises the administration of other therapeutic and/or preventive treatments.

For the purposes of the present invention, the term“pharmaceutically effective amount” shall be understood as an amount that provides a therapeutic effect without causing unacceptable toxic effects on the patient. The effective amount or dose of the medicament depends on the compound and the condition or disease treated, and for example the age, weight and clinical condition of the treated patient, the form of administration, the clinical history of the patient, the seriousness of the disease and the potency of the compound administered.

Additionally, the compounds of formula (I) and compositions comprising the same, disclosed in present description may also be administered together, prior to, or subsequently to another therapeutic and/or preventive treatments. Said other therapeutic and/or preventive treatment may be an additional therapy or, an additional active compound or therapeutic ingredient. Preferably said additional therapy is radiotherapy, immunotherapy or chemotherapy.

In a preferred embodiment, the compounds of formula (I) or the pharmaceutical composition comprising the same are administered together with, prior to or subsequently to a radiotherapeutic treatment, a chemotherapeutic treatment or an immunotherapeutic treatment.

The compositions for use according to the present invention can comprise one or more compounds of formula (I), as well as one or more active compounds. For the purposes of the present invention, active compound shall be understood as a chemical entity or molecule that has therapeutic effects when administered to a human being or an animal.

Accordingly, in one embodiment, the pharmaceutical compositions disclosed comprise at least one additional active compound or therapeutic ingredient.

In another embodiment the compounds of formula (I) are administered together with, prior to or subsequently to at least one additional active compound or therapeutic ingredient.

Said additional active compound or therapeutic ingredient provides additive or synergistic biological activities. For the purposes of present description, the terms“active compound” or“therapeutic ingredient” should be taken as synonyms and mean a chemical or biological entity which exerts therapeutic effects when administered to human or animal beings. Said active compound or therapeutic ingredient exerts therapeutic effects when administered to human or animal beings, and it may be a cell therapy, a small molecule therapy, a immunotherapy, radiotherapy, among others. Preferably said active compound or therapy is a chemotherapeutic agent, a cell therapy or an immunotherapeutic agent.

In a preferred embodiment said additional active compound or therapeutic agent is selected from the group consisting of a CAR-T cell therapy, a CAR-NK cell therapy, a monoclonal antibody, a HIF pathway inhibitor and a chemotherapeutic agent. Said additional active compound or additional therapy may be administered at the same time, prior to, subsequently to, or at a different moment than the compound of formula (I) for use according to present invention. Preferably, said chemotherapeutic agent is selected from the group consisting of platinum-based antineoplastic agents, anti-mitotic chemotherapeutic agents, a poly adenosine diphosphate ribose polymerase (PARP) inhibitor, type I topoisomerase inhibitors, type II topoisomerase inhibitors, epothilones, cycloskeletal disruptors, alkylating agents, epothilones, histone deacetylase inhibitors, kinase inhibitors, antifolates, kinase inhibitors, peptide antibiotics, retinoids, vinca alkaloids and thymidylate synthase inhibitors. More preferably, the chemotherapeutic agent is selected from the group consisting of cyclophosphamide, ifosfamide, busulfan, temozolomide, mechlorethamine, chlorambucil, melphalan, dacarbazine, daunorubicin, doxorubicin, daunorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin, paclitaxel, docetaxel, abraxane, taxotere, epothilone, vorinostat, romidepsin, irinotecan, topotecan, camptothecin, exatecan, lurtotecan, etoposide, teniposide, tafluposide, bortezomib, erlotinib, gefitinib, imatinib, vemurafenib, vismodegib, azacitadine, azathioprine, capecitabine, cytarabine, cladribine, fludarabine, doxifluridine, fluorouracil, gemcitabine, hydroxyurea, mercaptopurine, methotrexate, pemetrexed, tioguanine, bleomycin, actinomycin, carboplatin, cisplatin, oxaliplatin, tretinoin, alitretinoin, bexarotene, vinblastine, vincristine, vindesine and vinorelbine.

In yet another preferred embodiment said additional active ingredient is a monoclonal antibody. Preferably said monoclonal antibody is selected mogamulizumab, blinatumomab, ibritumomab, obinutuzumab, ofatumumab, rituximab, tositumomab, inotuzumab, moxetumomab, brentuximab, gemtuzumab, daratumumab, isatuximab, alemtuzumab, polatuzumab, cetuximab, necitumumab, nimotuzumab, panitumumab, catumaxomab, burosumab, dinutuximab, pertuzumab, trastuzumab, ertumaxomab, mepolizumab, siltuximab, enfortumab, etaracizumab, racotumomab, bevacizumab, denosumab, elotuzumab, olaratumab, ramucirumab, bermekimab, labetuzumab, pemtumomab, tacatuzumab, pembrolizumab, nivolumab, cemiplimab, atezolizumab, avelumab, durvalumab, ipilimumab or any other immunotherapeutic agent.

A preferred embodiment refers to a pharmaceutical composition for use according to the present invention, comprising a compound of formula (I) wherein:

- Ri is -COOH;

R2 is -CH2-; and

R3 is selected from:

a phenyl group having one OH substituent or having one or more substituents independently selected from the group consisting of: halogen, C1-C6 alkyl, -OR ₆ , - COOH and -COOR ₆ , wherein R ₆ is a C1-C6 alkyl group;

or

an unsubstituted 1 H-indol-3-yl group or a 1 H-indol-3-yl group having one or more substituents independently selected from the group consisting of: OH, halogen, C1-C6 alkyl, -OR ₆ , -COOH and -COOR ₆ , wherein R ₆ is an C1-C6 alkyl group;

and the compound has formula (II):

A more preferred embodiment refers to a pharmaceutical composition for use according to the present invention, comprising a compound of formula (II) wherein R3 is an unsubstituted 1 H-indol-3-yl group, and the compound of formula (II) is tryptophan-betaxanthin:

tryptophan-betaxanthin

Another preferred embodiment relates to a pharmaceutical composition for use according to the present invention, comprising a compound of formula (I) wherein:

- Ri is H;

R2 is -CH2-; and

R3 is selected from:

an unsubstituted phenyl group or a phenyl group having one OH substituent or having one or more substituents independently selected from the group consisting of: halogen, C1-C6 alkyl, -OR ₆ , -COOH and -COOR ₆ , wherein R ₆ is a C1-C6 alkyl group;

or

an unsubstituted 1 H-indol-3-yl group or a 1 H-indol-3-yl group having one or more substituents independently selected from the group consisting of: OH, halogen, C1-C6 alkyl, -OR ₆ , -COOH and -COOR ₆ , wherein R ₆ is an C1-C6 alkyl group;

and the compound has formula (III):

A more preferred embodiment refers to a pharmaceutical composition for use according to the present invention, comprising a compound of formula (III) wherein R ₃ is an unsubstituted phenyl group, and the compound of formula (III) is phenylethylamine-betaxanthin:

phenylethylamine-betaxanthin

The compositions for use according to the present invention include, together with the compounds described in the present invention, at least one pharmaceutically acceptable excipient which can be, among others, a carrier or diluent.

Said compositions can be included in capsules, pills, sachets or envelopes, or in any other presentation.

Conventional techniques for preparing pharmaceutical compositions can be used to prepare said compositions. For example, the compound of interest can be mixed with a carrier or diluted in a carrier or included in a carrier in the form of an ampoule, capsule, pill, envelope, sachet or other container. When the carrier acts as a solvent it may be solid, semi-solid, or liquid, and act as an excipient or medium for said active compound. The compound of interest can be adsorbed in a solid granular medium. Some examples of suitable vehicles are water, saline solutions, alcohols, polyethylene glycols, poyihydroxyethoxylated castor oil, peanut oil, olive oil, lactose, terra alba, saccharose, cyclodextrins, amylose, magnesium stearate, talcum, gelatin, agar, pectin, acacia, stearic acid, cellulose alkyl ethers, silicic acid, fatty acids, fatty acid amines, fatty acid monoglycerids and diglycerids, fatty esters of pentaerythrol, polyethylene, hydroxymethylcellulose and polyvinylpirrolidone. Likewise, the carrier can include controlled release materials known in the prior art, such as glyceryl monostearate or distearate, alone or mixed with a wax. The formulations can also include wetting agents, emulsifiers, suspension agents, preservatives, sweeteners or flavourers. The compositions can be formulated to provide a quick, prolonged or delayed release of the active agent after administration to the patient using known methods in the prior art.

The pharmaceutical compositions can be sterilized and mixed, if desired, with additional agents, emulsifiers, salt to influence osmotic pressure, buffers, and/or colorants that do not react adversely with the active compounds.

One embodiment refers to the form of administration, which can be any that effectively transports the compound of interest to the desired place of action, such as oral, rectal, or parenteral, for example, subcutaneous, intravenous, intraurethral, intramuscular, intranasal or as an ophthalmic solution.

Preferably, the pharmaceutical composition for use according to the present invention is a pill, an injectable solution or an ophthalmic solution.

For oral administration, both solid and liquid dosing forms can be prepared. To prepare solid forms such as pills, the compound of interest is mixed in a formulation with other conventional ingredients such as talcum, magnesium stearate, bicalcium phosphate, aluminium and magnesium silicate, starch, lactose, acacia, methylcellulose and functionally similar materials as pharmaceutical carriers and diluents.

The capsules can be prepared by mixing the compound of interest with a pharmaceutically inert solvent and filling the mixture in a hard gelatin with the suitable size. Soft capsules are prepared with encapsulation machines for suspensions of the compound of interest with an acceptable vegetable oil, light paraffin or inert oil. Liquid dosing forms can also be prepared, such as syrups, elixirs and suspensions. Water-soluble forms can be dissolved in an aqueous medium with sugar, flavouring aromas and preservatives to form a syrup. Elixirs are prepared using a hydroalcoholic carrier (such as ethanol) with suitable sweeteners such as sugar or saccharin, together with flavouring aromas. Suspensions can be prepared with an aqueous carrier and a suspension agent such as acacia, tragacanth, methylcellulose and the like.

For nasal administration, the preparation can contain the compound of interest dissolved or suspended in a liquid carrier, particularly an aqueous carrier, for application as an aerosol. The vehicle can contain additives such as solubilizing agents, for example, propyleneglycol, surfactants, absorption enhancers such as lecithin (phosphatidylcholine) or cyclodextrin, or preservatives such as parabens.

For ophthalmic applications, the compound of interest is formulated in solutions, suspensions and ointments appropriate for use in the eye. The concentrations are usually the same as in preparations for local use.

For parenteral application a person skilled in the art will find it obvious to use injectable solutions or suspensions, for intradermal, intramuscular, intravascular and subcutaneous use.

In addition to the compound of interest, the compositions can include other non-toxic pharmaceutically acceptable diluents and excipients, including carriers commonly used in pharmaceutical compositions normally used for humans or animals. The diluent is selected so that it does not affect the biological activity of the composition.

Examples of diluents especially used in injectable formulations are organic and inorganic salt solutions, Ringer's solution, dextrose solution and Hank’s solution. In addition, the compositions can include additives such as other excipients, adjuvating agents, non- therapeutic and non-immunogenic stabilizers and the like.

Examples of excipients that can be included in the formulation include but are not limited to cosolvents, surfactants, oils, wetting agents, emollients, preservatives, stabilizers and antioxidants. Any physiologically acceptable buffer can be used, such as Tris or phosphate buffers. Effective amounts of diluents, or additives or excipients are those that are effective for obtaining a pharmaceutically acceptable formulation with regard to solubility and biological activity.

Another embodiment relates to the dosing regimen. The term unit dose refers to physically discrete units suitable as unit doses for an individual, such as a mammal, human, dog, cat, rodent, etc. where each unit contains a specified amount of active material calculated to have the appropriate therapeutic effect in association with the appropriate diluent, carrier or support.

A preferred embodiment relates to a pharmaceutical composition for use according to the present invention, where said composition is administered in an oral, intramuscular, intravenous, subcutaneous, inhalatory, transdermal, nasal, ophthalmic, otic, topical, rectal or vaginal manner.

Since the compounds of formula (I) can also have cancer prevention effects and be used as chemopreventive agents, they can also form part of nutraceutic or food compositions together with other compounds that can also be used in the prevention of cancer.

Specifically, the compounds of formula (I) can also be used as chemopreventive agents, or to improve the health of oncological patients.

The term “chemopreventive agent” relates to a compound or substance which when administered delays or prevents the appearance of cancer.

Thus, the present invention also relates to a compound of formula (I), or an enantiomer, diastereomer or E/Z isomer thereof, more preferably to the use of a compound of formula

(II) or a compound of formula (III), even more preferably to the use of tryptophan- betaxanthin or of phenylethylamine-betaxanthin, for use as a chemopreventive agent, or to improve the health of oncological patients.

Another aspect of present invention refers to a method to delay or prevent the appearance of cancer in a subject, wherein said method comprises administering a nutraceutically effective amount of a compound of formula (I), or of an enantiomer, diastereomer or E/Z isomer thereof, more preferably of a compound of formula (II) or of a compound of formula

(III), even more preferably of tryptophan-betaxanthin or phenylethylamine-betaxanthin, or of a nutraceutical or food composition comprising the same, to said subject.

For the purposes of the present invention, the term“nutraceutically effective amount” shall be understood as an amount that provides a nutraceutic effect without causing unacceptable toxic effects on the patient.

Thus, another aspect of the invention refers to the use of a nutraceutical or food composition comprising a compound of formula (I), or an enantiomer, diastereomer or E/Z isomer thereof:

wherein Ri is H, -COOH, or -COOR ₆ , wherein R ₆ is a C ₁ -C ₆ alkyl group;

R ₂ is a C ₁ -C ₁₀ unsubstituted alkanediyl group or a C ₁ -C ₁₀ alkanediyl group with one or more substituents independently selected from the group consisting of: halogen, C ₁ -C ₆ alkyl, -R ₄ -COR ₅ and -R ₄ -COOR ₅ , wherein R ₄ is a C ₁ -C ₆ alkanediyl group and R ₅ is -H or a C ₁ -C ₆ alkyl group;

R ₃ is selected from:

an unsubstituted phenyl group or a phenyl group having one OH substituent or having one or more substituents independently selected from the group consisting of: halogen, C ₁ -C ₆ alkyl, -OR ₆ , -COOH and -COOR ₆ , wherein R ₆ is a C ₁ -C ₆ alkyl group;

or

an unsubstituted 1 H-indol-3-yl group or a 1 H-indol-3-yl group having one or more substituents independently selected from the group consisting of: OH, halogen, C ₁ -C ₆ alkyl, -OR ₆ , -COOH and -COOR ₆ , wherein R ₆ is an C ₁ -C ₆ alkyl group;

and wherein when R ₃ is an unsubstituted phenyl group, Ri is H;

and at least one acceptable food excipient; for the prevention of cancer or as a chemopreventive agent, for delaying or preventing the appearance of cancer.

The term“acceptable food excipient” relates in the present invention to a carrier or diluent.

For purposes of the present invention, a nutraceutical composition is understood as a food composition to be ingested separately or together with food and which has a medicinal effect on human health.

In a preferred embodiment the nutraceutical composition for use according to the invention comprises a compound of formula (I) wherein:

- Ri is -COOH;

R2 is -CH2-; and

R ₃ is selected from:

a phenyl group having one OH substituent or with one or more substituents independently selected from the group consisting of: halogen, C ₁ -C ₆ alkyl, -OR ₆ , - COOH and -COOR ₆ , wherein R ₆ is a C ₁ -C ₆ alkyl group;

or

an unsubstituted 1 H-indol-3-yl group or a 1 H-indol-3-yl group having one or more substituents independently selected from the group consisting of: OH, halogen, C ₁ -C ₆ alkyl, -OR ₆ , -COOH and -COOR ₆ , wherein R ₆ is an C ₁ -C ₆ alkyl group;

and the compound has formula (II):

In a more preferred embodiment, the nutraceutical composition for use according to the present invention comprises a compound of formula (II) wherein R3 is an unsubstituted 1 H- indol-3-yl group, and the compound of formula (II) is tryptophan-betaxanthin:

In another preferred embodiment the nutraceutical composition for use according to the invention comprises a compound of formula (I) wherein:

- Ri is H;

R2 is -CH2-; and

R3 is selected from:

an unsubstituted phenyl group or a phenyl group having one OH substituent or with one or more substituents independently selected from the group consisting of: halogen, C1-C6 alkyl, -OR ₆ , -COOH and -COOR ₆ , wherein R ₆ is a C1-C6 alkyl group;

or

an unsubstituted 1 H-indol-3-yl group or a 1 H-indol-3-yl group having one or more substituents independently selected from the group consisting of: OH, halogen, C1-C6 alkyl, -OR ₆ , -COOH and -COOR ₆ , wherein R ₆ is an C1-C6 alkyl group;

and the compound has formula (III):

In another more preferred embodiment, the nutraceutical composition for use according to the present invention comprises a compound of formula (III) wherein R3 is an unsubstituted phenyl group, and the compound of formula (III) is phenylethylamine-betaxanthin:

phenylethylamine-betaxanthin

In another preferred embodiment, the nutraceutical composition for use according to the invention is incorporated in a food preparation.

The nutraceutical composition for use according to the invention can also be included in a variety of food preparations, such as dairy products: yoghurt, quark, cheese (such as cottage cheese, cream, processed, soft and hard cheese), fermented milk, powdered milk, a fermented dairy product, ice cream, a product based on fermented cereal, milk-based powder, drinks, and pet food.

The term“food preparations” is used here in its broadest sense, including any type of product, in any presentation form, that can be ingested by a human or an animal, except for pharmaceutical and veterinarian products. Examples of other food preparations are meat products (such as pates, sausages, salami or spreadable meat), spreadable chocolate, fillers (such as truffles, cream) and cake icing, chocolate, sweets (such as caramel, fondant or toffee), baked products (pastries, biscuits), sauces and soups, fruit juices and coffee whitener. Food products of particular interest include food supplements and baby formulas.

The nutraceutical composition for use according to the invention can also be used as an ingredient in other food products. Consequently, in another aspect of the invention food products are provided that contain the composition of the invention as well as appropriate amounts of edible ingredients. Preferably, the nutraceutical composition for use according to the invention is a food supplement. For the purpose of the present invention, the term “food supplement” refers to the fraction of the food that is used to complement human or animal food. If the nutraceutical composition for use according to the invention is used as a food supplement, it can be administered as such or can be mixed with a suitable potable liquid, such as water, yoghurt, milk or fruit juice, or can be mixed with solid or liquid food. In this context, the food supplement can be in the form of tablets, pills, capsules, granules, powders, suspensions, sachets, pastilles, sweets, bars, syrups and corresponding forms for administration, generally as unit doses.

EXAMPLES

The examples described below are for purposes of illustration only and are not meant to limit the scope of the present invention.

Example 1 : Biotechnological production of compounds of formula (I)

The compounds of formula (I) were obtained using the method published by Guerrero-Rubio et al. , 2019 (Microbial Biotechnology. DOI: 10.11 11/1751-7915.13452).

Betalamic acid was produced by a novel and efficient DODA enzyme (4,5-DOPA-extradiol- dioxygenase) of Gluconacetobacter and subsequently condensed with an excess of the respective amine or individual amino acids in cell factories to obtain the betaxanthins. Four betalains were obtained having a structure without net positive charge. To this end the cell cultures containing the DODA enzyme, distilled water, 7,6 mM L-Dopa (L-3,4- dihydroxiphenylalanine), 15 mM sodium ascorbate and additionally the 38 mM L- tryptophan, L-phenylethylamine and L-phenylalanine amino acids or amines were added to a flask to obtain the compounds of formula (I), L-tryptophan-betaxanthin and L- phenylethylamine-betaxanthin, as well as the compounds L-phenylalanine-betaxanthin, and dopaxanthin. All of said steps under stirring and at 20 °C for 48 h.

These betalains having an imine group were purified to homogeneity by anion exchange chromatography and solid phase extraction, following a previously described method (Garcia-Herrero, Escribano & Garcia-Carmona, Planta, 2010. DOI: 10.1007/s00425-010- 1 191-0; Guerrero-Rubio et al, Microbial Biotechnology, 2019. DOI: 10.1 11 1/1751- 7915.13452 2019).

Example 2: In vitro assay of antioxidant capacity.

The antioxidant capacity of each molecule was evaluated by the ABTS method. This technique indicates whether an antioxidant compound can reduce the ABTS ⁺ radical to ABTS expressed in TEAC (Trolox equivalent antioxidant capacity). As shown in Figure 3, dopaxanthin has the highest antioxidant capacity in vitro (6.8 TEAC), as we expected due to the presence of the catecholic substructure (Gandia-Herrero et al., Journal of Natural Products, 2009. DOI: 10.1021/np900131 r; Gandia-Herrero et al., Planta, 2010. DOI: 10.1007/s00425-010-1 191-0). The values were also determined for tryptophan-betaxanthin (5.8 TEAC) phenylalanine-betaxanthin (2.6 TEAC) and phenylethylamine-betaxanthin (2.5 TEAC), which have an antioxidant capacity similar to that of betalamic acid (2.7 TEAC). Therefore, the results show that the molecule having the greatest antioxidant capacity is dopaxanthin.

Example 3. In vivo assay of antioxidant capacity.

The capacity to eliminate free radicals in vivo was studied using a TJ375 strain of the C. elegans nematode. This strain has fused the heat shock protein HSP-16,2 to the green fluorescent protein GFP, which accumulates in the pharynx of the animals when the animal is subjected to oxidative stress (Figures 4 B-C), assessing the accumulation using fluorescence microscopy. Figures 4 A and C show that dopaxanthin is also the strongest antioxidant in vivo since it reduces the oxidative stress in the animal by 84 %. Phenylalanine-betaxanthin and phenylethylamine-betaxanthin are also capable of reducing oxidative stress in the animals substantially, as the accumulation of fluorescence is reduced by 68.9 % and 64.9 % respectively. Tryptophan-betaxanthin is the least effective molecule in reducing oxidative stress (23.2 %). Accordingly, tryptophan-betaxanthin is not a good antioxidant in vivo.

Example 4. In vivo assay of anti-tumour capacity.

Each pure betaxanthin was tested individually. The JK1466 mutant strain of C. elegans (tumour phenotype) was treated with 25 mM of each of the molecules obtained in a standard culture medium for C. elegans (Medium S). The treatment was carried out during four days of adulthood and on the fourth day the size of the tumours was analysed. Considering the results of the literature and accepting the result of the works that suggest a direct relationship between the antioxidant activity and anti-tumour or anti-cancer activity, it was expected that, since dopaxanthin is the molecule with the greatest antioxidant capacity of those tested, it would also be the most effective one for the treatment of tumours.

To examine the effect of pure betaxanthins on tumour growth, the sizes of the gonads were measured from the loop region to the proximal region (Figure 5), including the uterus region when it was full of tumour cells. None of the pigments showed a growth response in the size of the tumour.

Unexpectedly, the greatest effect on tumour reduction was obtained with tryptophan- betaxanthin, which reduced the tumour by 56.4 % taking as starting point the size of the gonad of the wild type animals (N2) (Figure 6), followed by phenylethylamine-betaxanthin which reduced the tumour by 27.7 % (Figure 6).

On the other hand, phenylalanine-betaxanthin and dopaxanthin did not have a significant effect of the size of the tumour of C. elegans (Figure 6) despite being a powerful antioxidant in vivo, as shown in Figure 3. These data show that tumour growth was significantly reduced in nematodes that were fed individually with phenylethylamine-betaxanthin and, especially, with tryptophan-betaxanthin. Therefore, the fact that a molecule features antioxidant and antiradical activity, as all betalains feature (including dopaxanthin and phenylalanine- betaxanthin), does not imply that it is an anti-tumour agent. It was therefore not foreseeable that the most active molecules for reducing tumours were tryptophan-betaxanthin and phenylethylamine-betaxanthin.

Example 5. Effect on the longevity of the C. elegans tumour model.

Of the four betalains with imine structure, two were selected due to their tumour growth inhibition effect, and therefore survival assays were conducted on the JK1466 strain of C. elegans with phenylethylamine-betaxanthin and tryptophan-betaxanthin. The mean lifespan analysis was performed on the automatic platform based on the Lifespan machine (Stroustrup et ai, Nature Methods, 2013. DOI: 10.1038/nmeth.2475). The results show a significant extension in the mean lifespan of C. elegans treated with phenylethylamine- betaxanthin, where mean survival time increased from 8.2 to 9.1 days (Figure 7 C and D). Thus, mean survival time increased by 1 1.4 %. Tryptophan-betaxanthin also significantly extended mean survival by 9.3%, from 8.2 to 8.9 days (Figure 7 A and B). All mean survival increases observed are statistically significant (p < 0.05). Therefore, phenylethylamine- betaxanthin and tryptophan-betaxanthin not only reduced tumour growth but also increased the lifetime of C. elegans in a significant manner.

The results obtained for the present invention show the efficacy of tryptophan-betaxanthin as an anti-tumour treatment. This result was not foreseeable, since the data obtained for antioxidant activity both in vitro and in vivo suggested that dopaxanthin would be the most effective anti-tumour treatment, as it has the greatest antioxidant activity. These results show that even though antioxidant capacity provides a generic effect on free radicals scavenging which can sometimes be beneficial, as described in the literature, effective tumour treatment is related to the specific structure of each molecule, as is the case for tryptophan-betaxanthin and phenylethylamine-betaxanthin.