Compositions comprising plant extracts and their uses in the treatment of inflammatory diseases of the gastrointestinal tract

Abstract

The present invention relates to a composition comprising : (i) dry extract of Vaccinium myrtillus L. fruits titrated from 5 to 20% in proanthocyanidins; (ii) dry extract of Ribes Nigrum fruits titrated from 5 to 10% in anthocyanins; (iii) dry extract of Vaccinium Macrocarpon Aiton fruits titrated from 0.1 to 20% in proanthocyanidins; (iv) dry extract of Aronia Melanocarpa fruits titrated from 0.5 to 5% in proanthocyanidins; (v) dry extract of Punica granatum L. fruits titrated from 5 to 15% in ellagic acid; (vi) dry extract from leaves or fruits of Olea Europaea L. titrated from 0.001 to 1% in oleuropein; and (vii) 90%-pure inulin. It is further related to its use as a medicament or as a food supplement, or as a food product, or for a food for special medical purposes, in particular for the treatment of an inflammatory disease, preferably an inflammatory bowel disease or malabsorption syndrome.

Description

[0001] "COMPOSITIONS COMPRISING PLANT EXTRACTS AND THEIR USES IN

[0002] THE TREATMENT OF INFLAMMATORY DISEASES OF THE GASTROINTESTINAL TRACT"

[0003] Cross-Reference to Related Applications

[0004] This Patent Application claims priority from Italian Patent Application No. 102024000012148 filed on May 28, 2024, the entire disclosure of which is incorporated herein by reference.

[0005] Technical Field of the Invention

[0006] The present invention relates to compositions comprising dry extracts of plant origin, comprising polyphenols, inulin and optionally collagen hydrolysate, and their uses in the pharmaceutical and nutraceutical field, in particular, in the treatment of inflammatory diseases of the gastrointestinal tract.

[0007] State of the Art

[0008] The family of inflammatory bowel diseases (IBD) , with particular reference to Crohn's disease (CD) and ulcerative colitis (UC) , comprise complex multifactorial diseases due to endogenous and genetic susceptibility, as well as external and environmental factors.

[0009] Although the exact etiology of Crohn's disease remains unknown, the key role of oxidative stress in the pathogenesis of the disease is widely recognised (Alemany-Cosme et al. 2021) . Oxidative stress, caused by an overabundance of reactive oxygen species (ROS) , i.e., free radicals, is present locally and systemically in patients with Crohn's disease and seems to be associated with the imbalanced immune response and dysbiosis associated with the disease (Alemany- Cosme et al . 2021 ) .

[0010] The intestinal tract is under continuous attack by the luminal microbes and oxidised compounds present in the diet , exposing it to recurrent oxidative changes ( Pathel & Stappenback 2013 ) . An imbalance in intestinal redox homeostasis damages intestinal epithelial cells and the permeable barrier, activating dys functional immune responses and reducing immune tolerance (Moret et al . 2014 , Iborra et al . 2011 ) . The intestinal cells negatively af fected by oxidation express an excessive reaction to antigens , which triggers the overproduction of reactive oxygen species (ROS ) , thus inducing a further deterioration of the intestinal mucosa . This leads to an aberrant response to luminal agents and the development of abnormal inflammatory and dys functional chronic immune responses (Beltran et al . 2010 ) , as well as alterations in oxidation-reduction reactions and the redox balance . An adequate dietary intake of antioxidants is therefore essential to maintain low intracellular levels of oxidative species , thus maintaining a correct gastrointestinal redox balance ( Pathel & Stappenback 2013 ) .

[0011] As is known, polyphenols are a family of natural antioxidants , which include about 5 , 000 organic molecules in which a plurality of phenolic groups are associated in generally complex structures and with high molecular weight .

[0012] Polyphenols are widely present in the plant kingdom, being products of the secondary metabolism of many plants .

[0013] Although it has been hypothesised, through in vitro experiments , that the biomedical ef fects of polyphenols can be extremely positive ( for example as antioxidants ) , a problem associated with the intake of polyphenols with the diet is their low bioavailability .

[0014] In the presence of speci fic nutritional needs , such as those of subj ects suf fering from chronic gastrointestinal diseases , it is necessary to apply speci fic nutritional regimens based on the food diet but also , when necessary, on foods for special medical purposes ( FSMP ) .

[0015] FSMPs are intended for patients with limited, disturbed or altered ability to ingest , digest , absorb, metabolise or eliminate common foods or certain nutrients or metabolites , or with other nutritional needs determined by clinical conditions and whose dietary management cannot be carried out exclusively with the modi fication of the normal diet . However, the progress of knowledge about the metabolism of nutrients in pathological conditions and the national and European regulatory framework, impose the need to have nutritional solutions specially formulated and usable by patients suf fering from IBD and designed so as to obtain synergistic actions between the components identi fied, even with a low concentration of active ingredients .

[0016] Object and Summary of the Invention

[0017] With the present invention, a composition comprising 6 dry plant extracts , inulin and optionally collagen hydrolysate was surprisingly found, which possess antioxidant ef fects even at low percentages of active ingredients .

[0018] In particular, according to the present invention, a composition is provided according to claim 1 and its uses according to claims 5-7 and 10 .

[0019] Brief Description of the Drawings In order to better understand the present invention, some embodiments thereof will now be described, by way of mere non-limiting example, with reference to the accompanying drawings, wherein:

[0020] - Figure 1 indicates the values expressed as a percentage related DCF fluorescence following 24 h treatment of CaCo-2 cells with the extract compositions as indicated in the graph. TBH: tert-butyl-hydroperoxide . The concentrations indicated refer to pg / mL (n=3, n=5 for Ctrl- and Ctrl+) . * p<0.05 ** p<0.01 and *** p< 0.001 towards positive control (TBH) .

[0021] - Figure 2 shows the values expressed as a percentage related to the inhibition of DCF fluorescence following 24 h treatment of CaCo-2 cells with the extract compositions as indicated in the graph (n=3) . The concentrations indicated refer to pg / mL.

[0022] Preferred Embodiments of the Invention

[0023] The subject matter of the present invention is a composition comprising:

[0024] (i) dry extract of Vaccinium myrtillus L. fruits titrated from 5 to 20% in proanthocyanidins;

[0025] (ii) dry extract of Ribes Nigrum fruits titrated from 5 to 10% in anthocyanins;

[0026] (iii) dry extract of Vaccinium Macrocarpon Aiton fruits titrated from 0.1 to 20% in proanthocyanidins;

[0027] (iv) dry extract of Aronia Melanocarpa fruits titrated from 0.5 to 5% in proanthocyanidins;

[0028] (v) dry extract of Punica granatum L. fruits titrated from 5 to 15% in ellagic acid;

[0029] (vi) dry extract from leaves or fruits of Olea Europaea L. titrated from 0.001 to 1% in oleuropein; and

[0030] (vii) 90%-pure inulin; wherein:

[0031] (i) the dry extract of Vaccinium myrtillus L. fruits is comprised between 10 and 20%; preferably comprised between 12 and 17%; more preferably comprised between 13 and 15%;

[0032] (ii) the dry extract of Ribes Nigrum fruits is comprised between 10 and 20%; preferably comprised between 12 and 17%; more preferably comprised between 13 and 15%;

[0033] (iii) the dry extract of Vaccinium Macrocarpon Aiton fruits is comprised between 10 and 20%; preferably comprised between 12 and 17%; more preferably comprised between 13 and 15%;

[0034] (iv) the dry extract of Punica granatum L. fruits is comprised between 10 and 20%; preferably comprised between 12 and 17%; more preferably comprised between 13 and 15%;

[0035] (v) the dry extract of Aronia Melanocarpa fruits is comprised between 10 and 20%; preferably comprised between 12 and 17%; more preferably comprised between 13 and 15%;

[0036] (vi) the dry extract from leaves or fruits of Olea Europaea L. is comprised between 10 and 20%; preferably comprised between 12 and 17%; more preferably comprised between 13 and 15%;

[0037] (vii) the inulin is comprised between 10 and 20%; preferably comprised between 12 and 17%; more preferably comprised between 13 and 15%; the quantities being expressed as percentages by weight with respect to the total weight of the components i) -vii) .

[0038] In the present invention the term(s) "proanthocyanidin ( s ) " and proanthocyanin ( s ) " are to be understood as synonyms; the reference to one or the other is therefore indifferent.

[0039] The composition, rich in polyphenols including proanthocyanidins, anthocyanins, ellagic acid and oleuropein, acts on different molecular mechanisms that modulate gene expression and post-transcriptional patterns responsible for controlling inflammation, oxidative stress, immune response, carcinogenesis, angiogenesis and apoptosis.

[0040] The effects of such modulations on inflammatory diseases, both acute and chronic, are particularly important, in particular on inflammatory bowel diseases, thanks to the eubiotic action carried out on the microbiota by the presence of inulin. In fact, inulin acts as a prebiotic, enhancing the modulating effects on the intestinal microbiota exerted by the mixture of polyphenols contained in the extracts. Therefore, inulin promotes the effects on the microbiota, but does not act directly at the systemic level (cardiac, endothelial) as polyphenols instead do .

[0041] In a preferred embodiment, the composition according to the invention can have the following titrations of the active ingredients :

[0042] (i) the dry extract of Vaccinium myrtillus L. fruits is titrated at 10% in proanthocyanidins;

[0043] (ii) the dry extract of Ribes Nigrum fruits is titrated at 7% in anthocyanins;

[0044] (iii) the dry extract of Vaccinium Macrocarpon Aiton fruits is titrated at 1% in proanthocyanidins;

[0045] (iv) the dry extract of Aronia Melanocarpa fruits is titrated at 2% in proanthocyanidins; (v) the dry extract of Punica granatum L. fruits is titrated at 10% in ellagic acid;

[0046] (vi) the dry extract from leaves or fruits of Olea Europaea L. is titrated at 0.1% in oleuropein.

[0047] In an alternatively embodiment, the composition which is the subject matter of the present invention can have the following titrations of active ingredients:

[0048] (i) the dry extract of Vaccinium myrtillus L. fruits is titrated at 10% in proanthocyanidins;

[0049] (ii) the dry extract of Ribes Nigrum fruits is titrated at 7% in anthocyanins;

[0050] (iii) the dry extract of Vaccinium Macrocarpon Alton fruits is titrated at 10% in proanthocyanidins;

[0051] (iv) the dry extract of Aronia Melanocarpa fruits is titrated at 2% in proanthocyanidins;

[0052] (v) the dry extract of Punica granatum L. fruits is titrated at 10% in ellagic acid;

[0053] (vi) the dry extract from leaves or fruits of Olea Europaea L. is titrated at 0.1% in oleuropein.

[0054] Advantageously, despite the low concentrations of the individual active ingredients, the composition of the invention showed a significant antioxidant effect. The composition which is the subject matter of the present invention comprises the extract of Vaccinium myrtillus (1) fruits .

[0055] Vaccinium myrtillus (or bilberry) is a fruit shrub, classified as a plant that produces berries. Its fruits are small blue pseudo-berries. They are defined "pseudo-berries" since, although they have a berry-like appearance, ovaries, sepals, petals and stamens contribute to their formation. The extract used in the composition of the invention is generated from the pseudo-berries of Vaccinium myrtillus .

[0056] The extract of Vaccinium myrtillus fruits is preferably an extract titrated in proanthocyanidins (HPLC, Eur . Ph. method (d. e . ) ) .

[0057] The extract is obtained with an organic solvent, preferably comprising or alternatively consisting of an alcohol, even more preferably ethanol.

[0058] Said extract in dry form is obtained following the following steps:

[0059] (b.l) extracting Vaccinium myrtillus fruits with an organic solvent, preferably alcohol, even more preferably ethanol, to provide an extraction solution;

[0060] (b.2) filtering the extraction solution obtained from step (b.l) , to provide a filtered extraction solution and a waste plant material;

[0061] (b.3) absorbing the filtered extraction solution obtained from step (b.2) , to provide an absorbed extraction solution;

[0062] (b.4) separating the absorbed extraction solution obtained from step (b.3) , to provide a native extract and a residue / solvent;

[0063] (b.5) drying, grinding, mixing and sieving the native extract obtained from step (b.4) , to provide the dry extract and a further residue.

[0064] By way of example, an extract usable in the present composition is a powder with a particle size distribution such that not less than 90% by weight, preferably from 90% to 99% by weight, of the particles have a size less than 300 pm (and therefore have dimensions such as to pass through a 50 Mesh sieve) , more preferably with a loss on dryin ≤g 5.0%, and even more preferably with a total ash conten ≤t 5.0%.

[0065] The composition which is the subject matter of the present invention further comprises the extract of Ribes nigrum (ii) fruits.

[0066] Ribes nigrum (black currant or, less frequently, cassis, or Ribes nigrum L. ) is a plant in the family Grossulariaceae . Its fruits are in the form of globose black berries, rich in seeds, with the vestiges of the flower at the apex. Black currants differ from red currants not only in colour, but also in aroma, flavour and use of the fruits. The extract used in the composition of the present invention is obtained from the berries of Ribes nigrum.

[0067] Preferably, the extract of the Ribes nigrum fruits is an extract titrated in anthocyanins (HLPC method) .

[0068] Anthocyanins (or anthocyans) are compounds belonging to the flavonoid family, frequently used as water-soluble dyes. They are polyaromatic polyoxylated compounds capable of reacting with oxidising agents such as molecular oxygen or free radicals.

[0069] The extract is an extract obtained with a hydroalcoholic solvent, preferably a water-ethanol mixture. By way of example, the hydroalcoholic solvent could contain from 10% to 70vol. % of alcohol (preferably ethanol) , preferably from 20% to 60vol. %, even more preferably from 30% to 50vol. %, for example 40vol. %.

[0070] Said extract in dry form is obtained following the following steps:

[0071] (c.l) extracting the fruits of Ribes nigrum with a hydroalcoholic solvent, preferably a water-ethanol mixture, to provide an extraction solution;

[0072] (c.2) optionally centrifuging and / or filtering the extraction solution obtained from step (c.l) , to provide a centrifuged and / or filtered extraction solution and a waste plant material;

[0073] (c.3) concentrating the extraction solution obtained from step (c.l) or the centrifuged and / or filtered extraction solution obtained from step (c.2) , to provide a concentrated extraction solution and a solvent;

[0074] (c.4) preferably heat treating, and preferably adding ingredients, to the concentrated extraction solution obtained from step (c.3) to provide a native extract;

[0075] (c.5) preferably homogenising-mixing and / or preferably heat treating the concentrated extraction solution obtained from step (c.3) or the native extract obtained from step (c.4) , to provide a homogenised-mixed product and / or a heat- treated product;

[0076] (c.6) drying, preferably mixing with ingredients, and sieving the native extract obtained from step (c.4) or the homogenised-mixed product and / or the heat-treated product obtained from step (c.5) , to provide the dry extract (ii) and a residue.

[0077] The Ribes Nigrum extract is preferably in the form of a powder, preferably a powder with a density comprised between 400 g / 1 and 750 g / 1, more preferably comprised between 450 g / 1 and 650 g / 1. Even more preferably, a quantity by weight comprised between 80% and 100% (preferably comprised between 90% and 99%) of the particles of said powder has a mean particle size (or mean size distribution) comprised between 1 micrometre and 500 micrometres, preferably comprised between 10 micrometres and 300 micrometres .

[0078] Preferably, the extract in powder form has a total ash content ≤ 5.0%, and a loss on dryin ≤g 5.0%.

[0079] Even more preferably, the extract in powder form could contain excipients, preferably maltodextrins, even more preferably maltodextrins obtained from corn starch decomposition. Preferably, a weight ratio [extract powder (ii) ] : [maltodextrin] is comprised between 1:10 and 10:1, more preferably between 1:5 and 5:1, further preferably between 1 : 3 and 3:1.

[0080] The composition which is the subject matter of the present invention further comprises Vaccinium macrocarpon Alton ( iii ) .

[0081] Vaccinium macrocarpon Alton (American cranberry, or cranberry, or Vaccinium macrocarpon) is a fruit plant of the botanical family Ericaceae standing as a low woody bush, which produces red berries (fruits) from which the extract is obtained.

[0082] The extract is preferably obtained with a hydroalcoholic solvent, preferably a water-ethanol mixture. By way of example, a weight ratio water: ethanol in the water-ethanol mixture could be comprised between 80:1 and 1:80, preferably comprised between 50:1 and 1:50, even more preferably comprised between 25:1 and 1:25. the Vaccinium macrocarpon Alton extract in dry form is obtained following the following steps:

[0083] (a.l) extracting the fruits of Vaccinium macrocarpon Alton with the hydroalcoholic solvent, preferably the water- ethanol mixture, to provide an extraction solution; (a.2) concentrating the extraction solution obtained from step (a.l) , to provide a concentrated extraction solution;

[0084] (a.3) purifying the concentrated extraction solution obtained from step (a.2) , by means of a purification solution, preferably hydroalcoholic, even more preferably a water-ethanol mixture, to provide a purified extraction solution;

[0085] (a.4) concentrating the purified extraction solution obtained from step (a.3) , to provide a re-concentrated extraction solution;

[0086] (a.5) atomising the re-concentrated extraction solution obtained from step (a.4) , to provide the dry extract;

[0087] (a.6) preferably sieving, preferably weighing and preferably primarily packaging the dry extract obtained from step ( a .5 ) .

[0088] The extract of Vaccinium macrocarpon Alton fruits is an extract titrated in proanthocyanidins (Eur. Ph. method 6.0; 01 / 2008 : 12 20) .

[0089] Proanthocyanidins are molecular structures formed by oligomeric repeats of catechins and epicatechin, and their gallic acid esters, which lead to the formation of cyanidin when they are depolymerised under oxidative conditions. Proanthocyanidins are also called condensed tannins, as in the presence of strong acids they hydrolyse to provide anthocyanidins .

[0090] The extract of Vaccinium macrocarpon Alton fruits used in the present composition is preferably a water-soluble powder, more preferably a powder with a mean powder particle size (or mean powder particle size distribution) less than 250 micrometres. Preferably, such a water-soluble powder could contain excipients, preferably maltodextrins, even more preferably maltodextrins obtained from corn starch decomposition. Preferably, a water-soluble powder : maltodextrin weight ratio is comprised between 5:1 and 1:5, more preferably between 3:1 and 1:3.

[0091] Preferably, an aqueous solution of the extract of Vaccinium macrocarpon Alton fruits has a pH value comprised between 1.0 and 6.0, preferably between 2.0 and 4.5.

[0092] The composition which is the subject matter of the present invention comprises, in addition to the extract (i) , the extract (ii) and the extract (iii) , the extract of Aronia Melanocarpa (iv) fruits.

[0093] Aronia Melanocarpa is a medium-sized shrub, native to North America, which develops numerous erect and densely branched stems. Such a shrub generates small rounded, pendulous fruits, which become black when ripe, and which are edible.

[0094] The extract in dry form is obtained following a process comprising the following steps:

[0095] (e.l) extracting the Aronia Melanocarpa fruits with a hydroalcoholic solvent, preferably a water-ethanol mixture, to provide an extraction solution;

[0096] (e.2) centrifuging and / or filtering the extraction solution obtained from step (e.l) , to provide a centrifuged and / or filtered extraction solution (native extract) and a dry residue;

[0097] (e.3) controlling the dry residue and polycyclic aromatic hydrocarbon (PAG) content in the native extract obtained from step (e.2) ; (e.4) optionally adding maltodextrin and / or a different batch of native extract to the native extract obtained from step ( e .3 ) ;

[0098] (e.5) concentrating the native extract obtained from step (e.3) or step (e.4) , to provide a concentrated extraction solution and a solvent;

[0099] (e.6) atomising, and preferably adding auxiliary substances (preferably colloidal anhydrous silica) , to the concentrated extraction solution obtained from step (e.5) to provide a final product.

[0100] Preferably, the colloidal anhydrous silica added in step (e.6) is in a quantity comprised between 0.01% and 0.5% by weight with respect to the weight of the final product.

[0101] The extract is preferably in powder form. More preferably, a quantity by weight comprised between 80% and 100% (preferably comprised between 90% and 99%) of the particles of said powder has a mean particle size (or mean size distribution) comprised between 1 micrometre and 700 micrometres, preferably comprised between 10 micrometres and 500 micrometres (sieve method Eur . Ph.) , even more preferably a powder with a loss on drying ≤ 5.0%.

[0102] The extract of Aronia Melanocarpa fruits is preferably an extract titrated in proanthocyanidins (spectrophotometric method) in a percentage between 0.05% and 5%, more preferably comprised between 0.1% and 4%, even more preferably comprised between 0.5% and 3%, for example 2%.

[0103] Preferably, the extract is an extract obtained with a hydroalcoholic solvent, preferably a water-ethanol mixture. By way of example, the hydroalcoholic solvent could contain from 5% to 50vol. % of alcohol (preferably ethanol) , preferably from 10% to 45vol. % , more preferably from 20% to 40vol. % , for example 30vol. %.

[0104] The composition which is the subject matter of the present invention comprises, in addition to the extract (i) , the extract (ii) and the extract (iii) and the extract (iv) , the extract of Punica granatum (v) fruits.

[0105] Punica granatum (or pomegranate, or Punica granatum L. ) is a plant belonging to the family Punicaceae, native to a geographical region ranging from Iran to the Himalayan area of northern India, and which has been present since antiquity in the Caucasus and in the entire Mediterranean maquis. The fruit of the pomegranate tree is pomegranate, from which the extract (v) contained in the composition which is the subject matter of the present invention is obtained.

[0106] Preferably, the extract (v) is generated from the peel of pomegranate fruits, as it is rich in polyphenolic substances .

[0107] Said extract in dry form is obtained following the following steps:

[0108] (d.l) supplying and grinding Punica granatum fruits, to provide ground fruits;

[0109] (d.2) macerating the ground fruits obtained from step (d.l) with a hydroalcoholic solvent, preferably water- ethanol, for a time comprised between 10 minutes and 80 minutes, preferably between 20 minutes and 40 minutes, to provide a macerated liquid product;

[0110] (d.3) heating the macerated liquid obtained from step (d.2) to a temperature comprised between 50°C and 90°C, preferably comprised between 65°C and 75°C, at atmospheric pressure, for a time comprised between 30 minutes and 4 hours, preferably comprised between 1 hour and 2.5 hours, to provide a heated liquid product;

[0111] (d.4) centrifuging and / or filtering the heated liquid product obtained from step (d.3) , to provide a centrifuged and / or filtered extraction solution and a waste plant material ;

[0112] (d.5) concentrating the centrifuged and / or filtered extraction solution obtained from step (d.4) , preferably at atmospheric pressure, more preferably at a temperature comprised between 70°C and 90°C, even more preferably comprised between 75°C and 85°C, to provide a concentrated extraction solution;

[0113] (d.6) loading the concentrated extraction solution obtained from step (d.5) onto a chromatographic column, eluting with an organic solvent, preferably alcohol, more preferably ethanol, and collecting and concentrating the eluate, to provide a concentrated eluate liquid;

[0114] (d.7) atomising the concentrated eluted liquid obtained from step (d.6) to provide an atomised product;

[0115] (d.8) pulverising the atomised product obtained from step (d.7) to provide the dry extract (v) , and preferably sieving and / or mixing said dry extract (v) .

[0116] Preferably, in the maceration of step (d.2) following step (d.l) , a weight of hydroalcoholic solvent comprised between 3 and 15 times, preferably comprised between 5 and 10 times, even more preferably comprised between 7 and 9 times, the weight of the ground fruits obtained from step (d.l) is used.

[0117] Preferably, the waste plant material obtained from step (d.4) can be subjected again, one or more times, to the maceration step (d.2) and the subsequent heating step (d.3) , using fresh (not previously used) hydroalcoholic solvent in step (d.2) each time, so as to increase the extraction yield. The one or more centrifuged and / or filtered extraction solutions obtained from the repetition ( s ) of steps (d.2) , (d.3) and (d.4) may be fed (individually or after their union) as centrifuged and / or filtered extraction solution (s) at the next concentration of step (d.5) .

[0118] The extract of Punica granatum fruits is preferably an extract titrated in ellagic acid (CAS No. 476-66-4) (HLPC method) .

[0119] Ellagic acid is the dilactone of hexahydroxydiphenic acid, and shows antiproliferative and antioxidant properties in many in vitro models.

[0120] Preferably, the hydroalcoholic solvent used in step (d.2) contains from 40% to 95vol. % of alcohol (preferably ethanol) , more preferably from 50% to 90vol. %, further preferably from 60% to 80vol. %, for example 70vol. %.

[0121] Preferably, the extract (v) is in powder form. More preferably, a quantity by weight comprised between 80% and 100% (preferably comprised between 90% and 99%) of the particles of said powder has a mean particle size (or mean size distribution) such as to pass through an 80-mesh sieve.

[0122] Preferably, the extract in powder form has a total ash content ≤ 5.0%, and a loss on drying ≤ 5.0%.

[0123] Even more preferably, the extract in powder form could contain excipients, preferably maltodextrins, even more preferably maltodextrins obtained from corn starch decomposition. Preferably, a weight ratio [extract powder (v) ] : [maltodextrin] is comprised between 10:1 and 1:1, more preferably between 8:1 and 4:1.

[0124] The composition which is the subject matter of the invention further comprises a dry extract of Olea Europaea L . fruits ( vi ) .

[0125] The olive tree (Olea europaea L. , 1753) is a fruit tree that is presumed to be native to Asia Minor and Syria, because the spontaneous wild olive tree is very common in this region. Olives, their fruits, are used for the extraction of olive oil and, to a lesser extent, for direct use in food.

[0126] The extract (vi) is obtained from the leaves or fruits of such a tree and comprises tyrosol, or hydroxytyrosol, or oleuropein, or mixtures thereof.

[0127] The extract (vi) in dry form is obtained following a process comprising the following steps:

[0128] (f.l) extracting the Olea Europaea L. fruits with a hydroalcoholic solvent, preferably a water-ethanol mixture, to provide an extraction solution;

[0129] (f.2) centrifuging and / or filtering the extraction solution obtained from step (f.l) , to provide a centrifuged and / or filtered extraction solution (native extract) and a dry residue;

[0130] (f.3) controlling the dry residue and the oleuropein (or tyrosol, or hydroxytyrosol) content in the native extract obtained from step (f.2) ;

[0131] (f.4) optionally adding maltodextrin and / or a different batch of native extract to the native extract obtained from step (f.3) ;

[0132] (f.5) concentrating the native extract obtained from step (f.3) or step (f.4) , to provide a concentrated extraction solution and a solvent;

[0133] (f.6) atomising, and preferably adding auxiliary substances (preferably colloidal anhydrous silica) , to the concentrated extraction solution obtained from step (f.5) to provide a final product.

[0134] Preferably, the colloidal anhydrous silica added in step (f.6) is in a quantity comprised between 0.01% and 0.5% by weight with respect to the weight of the final product.

[0135] The extract (vi) is preferably in powder form. More preferably, a quantity by weight comprised between 80% and 100% (preferably comprised between 90% and 99%) of the particles of said powder has a mean particle size (or mean size distribution) comprised between 1 micrometre and 700 micrometres, preferably comprised between 10 micrometres and 500 micrometres (sieve method Eur . Ph.) , even more preferably a powder with a loss on drying ≤ 5.0%.

[0136] The dry extract from Olea Europaea L. fruit (vi) is preferably an extract titrated in oleuropein (or in tyrosol, or in hydroxytyrosol) .

[0137] Preferably, the extract (vi) is an extract obtained with a hydroalcoholic solvent, preferably a water-ethanol mixture. By way of example, the hydroalcoholic solvent could contain from 10% to 60vol. % of alcohol (preferably ethanol) , preferably from 20% to 55vol. %, more preferably from 30% to 50vol. %, for example 40vol. %.

[0138] The composition which is the subject matter of the present invention comprises, in addition to the extract (i) , the extract (ii) , the extract (iii) , the extract (iv) , the extract (v) , and the extract (vi) , inulin (vii) .

[0139] Inulin is a polymer of p-D-fructose (the monomers being linked by p-1 , 2-glycosidic bonds) , and is present mainly in Jerusalem artichoke tubers, chicory and black salsify roots (the latter, an herbaceous plant belonging to the family Asteraceae) .

[0140] An inulin usable in the present composition could have an average polymer chain length comprised between 5 and 20 monomers, preferably comprised between 6 and 15 monomers, even more preferably comprised between 8 and 13 monomers. Preferably, the inulin could be in powder form and, more preferably, could have an inulin content by weight comprised between 90% and 99%, and a total content by weight of fructose, glucose and sucrose comprised between 1% and 10%.

[0141] By way of example, the inulin is inulin CAS No. 9005- 80-5.

[0142] The inulin is preferably soluble inulin, i.e. with a solubility in water at 25°C comprised between 5 g / 1 and 200 g / 1.

[0143] In a preferred embodiment, the composition according to the invention further comprises viii) collagen hydrolysate.

[0144] Collagen is a protein composed by 19 amino acids, of which 8 are essential amino acids. The amino acid profile of collagen is unique and is not found in any other edible source .

[0145] In an aspect of the invention, the composition of the invention comprises a 1:1 mixture of whey proteins and collagen as a protein source, as the use of 2 protein sources allows having a balanced amino acid profile.

[0146] The selection of the correct protein substrate is decisive in the FSMP since it affects both its tolerability and the digestion, absorption and use of the product by the human body .

[0147] In the composition, a selection of proteins of high quality and high biological value was carried out , selecting proteins with a clinically irrelevant lactose concentration (<0 . 5 g / l O Og) as suggested by the ESPEN guidelines . In fact , said guidelines indicate that frequent food intolerances are detected in IBD patients and that lactase deficiency is prevalent in patients with proximal Crohn ' s disease . Whey proteins also contain all the essential and non-essential amino acids (EAA) and are rich in branched-chain amino acids (BCAA) , particularly leucine , a key amino acid for protein synthesis and a high cysteine content that participates in the synthesis of glutathione . Some clinical trials have also shown that whey proteins determine a greater postprandial stimulation of muscle protein anabolism and protein synthesis in general , with respect to other substrates such as caseins or soy proteins .

[0148] The composition of the invention comprises whey proteins and hydrolysed collagen proteins , preferably bovine collagen, whose absorption capacity is better than the acid casein commonly used in other products in the field .

[0149] In an aspect of the invention, the collagen hydrolysate is comprised between 5 and 20% ; preferably comprised between 5 and 15% ; more preferably comprised between 8 and 10% as a percentage by weight with respect to the total weight of the components i ) -viii ) .

[0150] Collagen hydrolysate , in addition to being a valid protein alternative , when included in the composition of the invention comprising the components from i ) to vii ) , has shown a surprising antioxidant ef fect . In an embodiment , the composition according to the invention further comprises at least one physiologically and / or pharmaceutically acceptable excipient .

[0151] In an aspect , the at least one excipient is a food additive such as silicon dioxide .

[0152] Advantageously, the composition according to the invention has been shown to be ef fective in improving the state of health o f a subj ect also from the nutritional point of view, decreasing oxidative stress .

[0153] Another obj ect of the present invention is the composition according to the invention for use as a medicament . Another obj ect of the present invention is the use of the composition as a food supplement , or as a food product , or for a food for special medical purposes ( FSMP ) . Said composition can further comprise a plurality of nutrients selected from the group comprising proteins , fats , vitamins , elements or minerals , and combinations thereof .

[0154] In an aspect of the invention, the use of the composition is for treating malabsorption syndrome .

[0155] In an alternative aspect of the invention, the use of the composition i s in particular for treating an inflammatory disease , preferably an inflammatory bowel disease .

[0156] In a preferred aspect , said inflammatory bowel disease is a chronic inflammatory bowel disease .

[0157] Within the scope of the invention, the term chronic inflammatory bowel disease is intended to mean chronic, permanent and recurrent diseases of the gastrointestinal tract , comprising phases of cyclical remission and exacerbation of the inflammatory process of the intestine .

[0158] Typically, these cyclical phases include malabsorption, hypercatabolism and reduced nutrient intake with possible malnutrition and vitamin and trace element deficiencies .

[0159] In a further preferred aspect , said chronic inflammatory bowel disease is Crohn ' s disease ( CD) or ulcerative colitis (UC ) .

[0160] In a further embodiment , the daily doses of the composition alone , excluding excipients , according to the invention are administered to a subj ect in the range of 25- 500 g / day .

[0161] Assuming 100% absorption of the composition, without metabolic interventions at the hepatic level , the presumed quantity in the blood, and therefore in contact with the intestinal mucosa, should correspond to 5 and 100 pg / mL, respectively .

[0162] Finally, it is clear that modi fications and variations can be made to the embodiments described and illustrated herein without thereby departing from the scope of protection of the present invention, as defined in the appended claims .

[0163] Examples

[0164] Example 1 - compositions

[0165] In the testing, the biological activities of compositions A and C were studied, in comparison with composition B :

[0166] A) New Extracts composition ( indicated with NE )

[0167] B ) Old extracts composition ( indicated as VE ) according to what is described in WO2021074913 .

[0168] C ) New Extracts + Collagen composition ( indicated as NE + Coll ) .

[0169] The compositions reported above are illustrated in Table 1 (NE composition) , Table 2 (VE composition) and Table (NE+Collagen composition) .

[0170] Table 1

[0171] (**) The ratio between the different extracts + inulin in the NE or VE mixture was 1:1: 1:1: 1:1: 1 and reflects the ratio between the different extracts in the finished product. The NE composition contains a quantity of titrated active ingredients equal to 4.3% w / w (weight / weight) ; while the VE composition contains a quantity of titrated active ingredients equal to 19.6% w / w.

[0172] In order to compare the same percentage (5%) of titrated active ingredients, a 4-fold dilution of the VE composition was carried out.

[0173] All the individual extracts were dissolved at the concentration of 10 mg / mL in the complete culture medium and diluted to the final concentrations directly in the culture plates .

[0174] The concentrations used in the 24 h pretreatment of the cells were 2.5, 5 and 100 pg / mL for each component in the NE and NE+Collagen compositions; 5 and 100 pg / mL for the VE composition .

[0175] By way of example, for the preparation of the tested mixtures, it is possible to proceed with the mixing, according to the proportions reported in Table 3, of 10 mg for all the extracts and inulin (total 70 mg) to which 7 mg of collagen (in fact present at 10% in the mixture, as reported in Table 3) is added.

[0176] Example 2 - materials and methods

[0177] Cell cultures

[0178] The cells used for this study are derived from an adenocarcinoma of the colon (CaCo-2, RRID: CVCL_0025) ; it is a cell line widely used in research as a model for intestinal epithelial tissue. The culture medium used is D-MEM High Glucose, supplemented with 10% foetal bovine serum (FBS) , 1 mM L-glutamine and antibiotics (100 lU / ml penicillin and 100 pg / ml streptomycin) .

[0179] Antioxidant assay

[0180] For the evaluation of the antioxidant activity of the 3 compositions, a method was used that determines the presence of ROS in the intracellular environment by means of a vital staining with dichlorofluorescein diacetate (DCFH- DA) (Eruslanov E, Kusmartsev S (2010) ) . Identification of ROS using oxidized DCFDA and flow-cytometry. Methods Mol Biol.; 594:57-72) . The latter is a non-polar molecule, capable of passing through the cell membrane and spreading easily inside cells, where it is hydrolysed by specific intracellular esterases to a non-f luorescent compound, dichlorofluorescein (DCFH) . In the presence of ROS, DCFH is oxidised to a highly fluorescent compound, 2,7- dichlorof luorescein (DCF) , which it emits at the wavelength of 535 nm. The fluorescence intensity is proportional to the quantity of intracellular ROS that were formed after treatments with oxidising agents. In 3 cm plates, 3.5xl05CaCo-2 cells were seeded and left in the incubator for 48 h, to allow adhesion and resumption of growth. They were then treated for 24 h with the compositions (VE at concentrations of 1.25, 5, 25 and 100 pg / mL, NE and NE+collagen at concentrations of 5 and 100 pg / mL) . At the end of the treatment, the medium was replaced with 3 mL of PBS containing 2 nM of DCFH-DA and the cells incubated for 30 minutes in the dark at 37°C and 5% CO2; after washing with PBS to remove the unabsorbed DCFHDA, the oxidising agent TBH (2.5 mM) was added in PBS for 40 min; finally, the cells were detached by scraper, centrifuged (3000 rpm for 2 minutes) and resuspended in 1 ml of PBS for flow cytometry reading. In parallel, a positive control treated with the oxidising agent TBH (Ctrl+) , a negative control (untreated cells, Ctrl-) and a sample free of DCFH-DA as "blank" were set up. The increase in fluorescence was determined with Attune NxT Acoustic Focusing flow cytometer (Thermo Lab Scientific) by mono-parametric analysis specific for the fluorescence emitted by DCF at an excitation length of 485 nm and emission length of 535 nm, respectively. The mean fluorescence intensity values obtained were expressed as a percentage with respect to the positive control.

[0181] Example 3 - Evaluation of antioxidant activity

[0182] The results were provided in graphical form and tables reporting the percentages of production (or inhibition) of intracellular ROS (DCFH-DA test) in CaCo-2 cells after pretreatment with the study compositions, with respect to those treated with TBH (positive control) and treated with the tested compounds (tests) . The values shown represent the average of three independent experiments.

[0183] The results are shown in Figure 1 in which the antioxidant activity, expressed as ROS levels, of the two VE samples, diluted 4 times, therefore at the same concentration of anthocyanins and ellagic acid, are compared with the compositions at the respective undiluted concentrations. In particular, the comparison is between the composition at 100 pg / mL of NE or NE+collagen and that at 25 pg / mL VE, and between 5 pg / mL of NE or NE+collagen and that at 1.25 pg / mL VE . The VE composition (25 pg / mL) determines a significant (p<0.05) increase of ROS equal to 37% with respect to the NE composition and 41% with respect to the NE+collagen composition. At the dose less than 5 pg / mL, the dilution of the VE composition (1.25 pg / mL) determines a further increase in ROS towards all compositions; with a significant increase of 49% only with respect to the NE+collagen composition (p<0.05) . In Figure 1, the symbols indicate respectively: • p<0.05 towards NE, NE+collagen and VE (100 pg / mL) ; ° p<0.05 towards NE+collagen (5 pg / mL) . Figure 2, on the other hand, and Table 4 report the percentages of inhibition of ROS formation induced by TBH after 24 h pretreatment by the compositions compared to the VE composition diluted 4 folds and evaluated in CaCo-2 cells. In Figure 2, the symbols indicate respectively: ° p<0.05 towards VE 1.25; • p<0.05 towards VE 25.

[0184] Table 4

[0185] From the experiments reported above it is therefore demonstrated that: • There is a reduction of ROS (and therefore an antioxidant effect) by all compositions with respect to the positive control (TBH) in CaCo-2 cells.

[0186] • The presence of collagen strengthens the antioxidant activity, especially at the dose of 5 pg / mL where the value tends to be significant if compared (p=0.07) with the same concentration of NE at the dose of 5 pg / mL.

[0187] • At the same concentration of the anthocyanin and ellagic acid content, the NE- and NE+collagen compositions have a significantly (p<0.05) higher antioxidant activity with respect to the VE composition.

Claims

CLAIMS1. Composition comprising:(i) dry extract of Vaccinium myrtillus L. fruits titrated from 5 to 20% in proanthocyanidins;(ii) dry extract of Ribes Nigrum fruits titrated from 5 to 10% in anthocyanins;(iii) dry extract of Vaccinium Macrocarpon Aiton fruits titrated from 0.1 to 20% in proanthocyanidins;(iv) dry extract of Aronia Melanocarpa fruits titrated from 0.5 to 5% in proanthocyanidins;(v) dry extract of Punica granatum L. fruits titrated from 5 to 15% in ellagic acid;(vi) dry extract from leaves or fruits of Olea Europaea L. titrated from 0.001 to 1% in oleuropein; and(vii) 90%-pure inulin; wherein :(i) the dry extract of Vaccinium myrtillus L. fruits is comprised between 10 and 20%; preferably comprised between 12 and 17%; more preferably comprised between 13 and 15%;(ii) the dry extract of Ribes Nigrum fruits is comprised between 10 and 20%; preferably comprised between 12 and 17%; more preferably comprised between 13 and 15%;(iii) the dry extract of Vaccinium Macrocarpon Aiton fruits is comprised between 10 and 20%; preferably comprised between 12 and 17%; more preferably comprised between 13 and 15%;(iv) the dry extract of Punica granatum L. fruits is comprised between 10 and 20%; preferably comprised between 12 and 17%; more preferably comprised between 13 and 15%;(v) the dry extract of Aronia Melanocarpa fruits iscomprised between 10 and 20%; preferably comprised between 12 and 17%; more preferably comprised between 13 and 15%;(vi) the dry extract from leaves or fruits of Olea Europaea L. is comprised between 10 and 20%; preferably comprised between 12 and 17%; more preferably comprised between 13 and 15%;(vii) the inulin is comprised between 10 and 20%; preferably comprised between 12 and 17%; more preferably comprised between 13 and 15%; the quantities being expressed as percentages by weight with respect to the total weight of the components i) -vii) .

2. Composition according to claim 1 further comprising viii) collagen hydrolysate.

3. Composition according to claim 2 wherein the collagen hydrolysate is comprised between 5 and 20%; preferably comprised between 5 and 15%; more preferably comprised between 8 and 10% as a percentage by weight with respect to the total weight of the components i)-viii) .

4. Composition according to any one of claims 1-3 further comprising at least one physiologically and / or pharmaceutically acceptable excipient.

5. Composition according to any one of claims 1-4 for use as a medicament.

6. Composition according to any one of claims 1-4 for use in the treatment of a malabsorption syndrome.

7. Composition according to any one of claims 1-4 for use in the treatment of an inflammatory bowel disease.

8. Composition for use according to claim 7 wherein said inflammatory bowel disease is a chronic inflammatory bowel disease.

9. Composition for use according to claim 8 wherein said chronic inflammatory bowel disease is Crohn's disease or ulcerative colitis.

10. Use of the composition according to any one of claims 1-4 as a food supplement, or as a food product, or for a food for special medical purposes.

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