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FIELD OF THE INVENTION

The present invention originates in the nutraceutical, pharmaceutical sectors, and relates to a mixture comprising a combination of selected polyphenols with inulin, and uses thereof in the medical field, in particular in the treatment or prevention of gastrointestinal inflammatory diseases and/or autoimmune diseases and related symptoms.

The present invention also relates to a composition comprising said mixture; and at least one physiologically and/or pharmaceutically acceptable excipient.

The present invention further relates to said composition comprising said mixture for use in the treatment of inflammatory diseases, more preferably acute and chronic inflammatory diseases, even more preferably for use in the treatment of inflammatory diseases selected from an inflammatory bowel disease or an autoimmune disease, in particular with an inflammatory component.

The present invention further relates to said composition comprising said mixture for use in the treatment of a condition or disorder or disease resulting from ischemia, preferably myocardial ischemia, or an alteration of the vascular endothelium function.

PRIOR ART

Polyphenols are a family of natural antioxidants, including about 5,000 organic molecules in which a plurality of phenolic groups are associated in generally complex and high molecular weight structures.

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

Although it was hypothesized, through in vitro experiments, that biomedical effects of polyphenols might be extremely positive (for example as antioxidants), a problem associated with the intake of polyphenols in the diet is their low bioavailability.

More precisely, polyphenols are not present in body fluids in their native form, but as metabolites (for example sulfated, methylated or glucuronated derivatives) due to metabolic transformation processes taking place in the intestine and liver.

The need is therefore felt to have a mixture rich in polyphenols, designed to achieve an increased bioavailability and to obtain synergistic actions of the identified components.

SUMMARY OF THE INVENTION

The Applicant, after a long and intense research and development activity, has developed a mixture of plant extracts containing polyphenols able to provide an adequate response to limitations, drawbacks and existing problems.

Therefore, the object of the present invention is a mixture (m) comprising or, alternatively, consisting of: (i) a Vaccinium macrocarpon fruit extract; (ii) a Vaccinium myrtillus fruit extract; (iii) a Ribes nigrum fruit extract; (iv) a Punica granatum fruit extract; (v) an Aronia Melanocarpa fruit extract; (vi) a mixture of polyphenols extracted from leaves or fruits of Olea Europaea L; (vii) inulin (fructose polymer) (in short, jointly, compounds (i)-(vii)), having the characteristics and/or uses as defined in the appended claims.

It is also an object of the present invention a composition (c) comprising said mixture (m), and at least one physiologically and/or pharmaceutically acceptable excipient, having the characteristics and/or uses in the medical or nutritional fields, as defined in the appended claims.

It is also an object of the present invention said mixture (m) or composition (c) for use in the treatment of inflammatory diseases, preferably acute and chronic inflammatory diseases, even more preferably for use in the treatment of inflammatory diseases selected from an inflammatory bowel disease or systemic inflammation, having the characteristics as defined in the appended claims.

A further object of the present invention is said composition (c) for use in the treatment of an autoimmune condition or disorder or disease, in particular with an associated inflammatory component or symptomatology, having the characteristics as defined in the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Preferred embodiments of the present invention will now be illustrated below, by way of non-limiting examples, with the aid of the attached tables, wherein:

- Figure 1 shows a flow diagram of a process for obtaining a Vaccinium macrocarpon fruit extract, in accordance with a possible embodiment;

- Figure 2 exemplifies a flow diagram of a process for obtaining a Vaccinium myrtillus fruit extract, in accordance with a possible embodiment;

- Figure 3 exemplifies a flow diagram of a process for obtaining a Ribes nigrum fruit extract, in accordance with a possible embodiment;

- Figure 4 exemplifies a flow diagram of a process for obtaining a Punica granatum fruit extract, in accordance with a possible embodiment;

- Figure 5 exemplifies a flow diagram of a process for obtaining an Aronia Melanocarpa fruit extract, in accordance with a possible embodiment;

- Figure 6 exemplifies a flow diagram of a process for obtaining an extract from leaves or fruits of Olea Europaea L, in accordance with a possible embodiment;

- Figure 7 shows bar graphs illustrating the results of Example 3, relating to the TNF- alpha ischemic marker gene expression data, after 24 hours of incubation with components (i) - (vii) of mixture (m).

In the positive control with cells treated with complete culture medium and recombinant TNF-alpha, a significant increase in TNF-alpha is observed. Data in Figure 7 show that the greatest reduction in TNF-alpha gene expression occurs with treatment using COMBINATION 1 , wherein all components i) - vii) of mixture (m) are present. There effect found is synergistic with respect to the other combinations tested;

- Figure 8 shows bar graphs illustrating the results of Example 3 relating to the inflammatory marker IL-8 gene expression after 24 hours of incubation with the compounds under analysis. The bar graphs show that in the positive control, comprising cells treated with complete culture medium and recombinant TNF-alpha, there is a significant reduction in IL-8. Figure 8 shows how the greatest increase in the SOD-2 gene expression occurs in the case of treatment with COMBINATION 1 , wherein all components i) - vii) of mixture (m) are present. There is a synergistic effect with respect to the other combinations tested.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with one aspect of the invention, the inventors of the present invention have selected - among the thousands of polyphenolic substances available in nature - the extracts from (i) to (vi) listed above as sources of polyphenols, combining them in the mixture (m) and adding inulin (vii) to this mixture (m). This combination of the biologically active components (i) - (vii) of the mixture modulates different biological or molecular mechanisms governing the regulation of the inflammation process, in particular at the level of the gastrointestinal tract, conferring a biological activity higher than what would be expected based on the sum of the activities of single active components (i)-(vii).

A mixture (m) is described herein comprising or, alternatively, consisting of:

(i) a Vaccinium macrocarpon fruit extract;

(ii) a Vaccinium myrtillus fruit extract;

(iii) a Ribes nigrum fruit extract;

(iv) a Punica granatum fruit extract;

(v) an Aronia Melanocarpa fruit extract;

(vi) a mixture of polyphenols extracted from leaves or fruits of Olea Europaea L;

(vii) inulin.

In accordance with another aspect, the present invention relates to a composition (c) comprising a mixture (m) as described herein and a pharmaceutically acceptable or edible excipient or vehicle and, advantageously, uses thereof in the treatment of inflammatory gastrointestinal diseases.

It has been observed that the biological mechanisms involved in the joint use of extracts (i) - (vi), especially when used in combination with inulin (vii), which exerts a direct action on intestinal eubiosis, preside over the control of local and systemic inflammation, oxidative stress, apoptosis, exerting an activity which favor the growth of eubiotic species within the intestinal microbiota and counteracting the onset of dysbiosis, one of the potential contributing causes of inflammatory bowel diseases and autoimmune diseases.

Inulin acts as a prebiotic, enhancing the modulating effects exerted by the mixture of polyphenols contained in the extracts (i) - (vi) on the intestinal microbiota. Inulin, therefore, favors the effects on the microbiota, but does not act directly at the systemic level (cardiac, endothelial) while polyphenols do.

The inventors have also found that compounds (i) - (vii) have immunomodulating effects by acting primarily on the intestine, where most of the immunocompetent cells are located and, consequently, at the systemic level. In accordance with one aspect, the present invention relates to a mixture (m) or a composition (c) as described herein for use in the treatment of an autoimmune disease.

Among polyphenols contained in the mixture (m), the co-presence of proanthocyanidins, anthocyanins, ellagic acid, oleuropein - preferably in the presence of phenolic alcohols and secoiridoid derivatives - act on different molecular mechanisms that modulate gene expression and post-transcriptional patterns in charge of controlling inflammation, oxidative stress, immune response, carcinogenesis, angiogenesis, and apoptosis.

The effects of these modulations are particularly important on inflammatory diseases, either with acute onset or chronic, in particular on inflammatory bowel diseases, thanks to the eubiotic action carried out on the microbiota by the presence of inulin (vii) in the mixture ( m), or on systemic inflammations of any origin that, in the event of chronicization, may result in a permanent organ damage, depending on their localization. Polyphenols contained in the mixture (m) modulate the pathophysiological mechanisms underlying inflammatory bowel diseases.

The synergistic effect of polyphenols contained in extracts (i) - (vi) and inulin (vii) present in the mixture (m) are largely due to a favorable modulation of the intestinal microbiota capable of regulating and increasing the biodiversity of bacterial species resident therein; these bacterial species, in fact, are able to modulate bioavailability and biotransformation of polyphenols in the mixture (m), increasing the local and systemic actions thereof. Precisely this bidirectional relationship between the polyphenols identified in the present mixture (m) and the bacterial species of the intestinal microbiota allows, inter alia, to inhibit a potential pathogens growth, through bacteriostatic and/or bactericidal activities, and on the other hand promotes a proliferation of eubiotic bacterial species.

The oxidative stress and/or inflammation control effect of the present mixture (m) is also expressed directly on the intestinal wall of the subject, reducing the possible radical damage on its cellular constituents, improving their integrity, and therefore maintaining their barrier function that opposes the penetration of toxic substances, bacterial derivatives, and products of incomplete enzymatic degradation of macro constituents present in the diet. In this way, the immunocompetent cells present in the submucosa have limited possibilities for activating the synthesis of inflammatory mediators, thus reducing the risk of triggering local inflammation with possible systemic manifestations.

In accordance with one aspect, the present invention relates to a mixture (m) or composition (c) as defined herein for use in the treatment of an inflammatory bowel disease in particular a chronic inflammatory bowel disease (MICI) or Inflammatory Bowel Disease (IBD).

Within the scope of the invention, the term “chronic inflammatory bowel disease” means chronic, permanent and relapsing diseases of the gastrointestinal tract, including cyclical remission and exacerbation phases of the inflammatory process of the intestine. Typically, malabsorption, hypercatabolism, and reduced intake of nutrients occur in these cyclical phases, with possible malnutrition, and vitamin and trace element deficiencies.

IBDs treatable with the mixture/composition comprise Crohn’s disease, a disease characterized by transmural inflammation that affects the intestinal wall throughout its thickness and “jump lesions”, located in any region of the gastrointestinal tract.

It was found that treatment with the mixture or composition of the invention reduces the symptoms of Crohn’s disease such as abdominal pain, diarrhea and extraintestinal manifestations, and also finds application in the treatment of ulcerative colitis.

One or more of extract (i), extract (ii), extract (iii), extract (iv), extract (v), extract (vi) are preferably a dry extract. Preferably, extracts (i) - (vi) are all dry extracts. Preferably, an [extract (i)] : [extract (iii)] : [extract (iv)] weight ratio is of about 1 :1 :1. An [extract (ii)] : [extract (i)] weight ratio preferably ranges from 4:1 to 1 :4, more preferably from 2:1 to 1 :2, even more preferably from 1.5:1 to 1 :1.

Preferably, an [extract (v)] : [extract (i)] weight ratio ranges from 20:1 to 1 :1 , preferably from 15:1 to 2:1 , even more preferably from 10:1 to 5:1.

Preferably, an [extract (vi)] : [extract (i)] weight ratio ranges from 15:1 to 1 :10, more preferably from 10:1 to 1 :1 , even more preferably from 8:1 to 2:1 .

Preferably, an [inulin (vii)] : [extracts (i)-(vi)] weight ration ranges from 10:1 to 1 :10, more preferably from 2:1 to 1 :5, even more preferably from 1 :1 to 1 :2.

Vaccinium macrocarpon (American cranberry, or large cranberry, or Vaccinium macrocarpon aiton) is a fruit plant of the Ericaceae botanical family of low woody shrub habit, producing red berries (fruits) from which the extract (i) is obtained. Extract (i) is an extract preferably obtained using a hydroalcoholic solvent, preferably a water-ethanol mixture. By way of example, a water : ethanol weight ratio in the water-ethanol mixture might range from 80:1 to 1 :80, preferably from 50:1 to 1 :50, even more preferably from 25:1 to 1 :25.

The amount by weight of Vaccinium macrocarpon fruit extract (i), with respect to the total weight of the mixture (m), preferably ranges from 1 % to 15%, more preferably from 1 .5% to 11 %, even more preferably from 2% to 9%, for example 4.6%.

The attached Figure 1 shows a flow diagram of a process for obtaining extract (i), in accordance with a possible embodiment. In accordance with such preferred process, the extract (i) in dry form is obtained as a result of the following steps: (a.1 ) extraction of Vaccinium macrocarpon fruits using the hydroalcoholic solvent, preferably the water-ethanol mixture, to give an extraction solution;

(a.2) concentration of the extraction solution obtained from step (a.1 ), to give a concentrated extraction solution;

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

(a.4) concentration of the purified extraction solution obtained from step (a.3), to give a reconcentrated extraction solution;

(a.5) atomization of the reconcentrated extraction solution obtained from step (a.4), to give the dry extract;

(a.6) preferably sieving, preferably weighing and preferably primary packaging of the dry extract obtained from step (a.5).

Preferably, the extraction of step (a.1 ) is carried out in an extraction tank, more preferably made of stainless steel.

The concentration of step (a.2) is preferably carried out in a concentrator (or evaporator), more preferably a single-effect one, even more preferably made of stainless steel.

The Vaccinium macrocarpon fruit extract (i) is preferably an extract titrated in proanthocyanidins (meth. Ph. Eur. 6.0; 01/2008 : 12 20) in a percentage ranging from 30% to 50%, preferably from 33% to 47%, more preferably from 35% to 45%, even more preferably from 38% to 42%, for example 38%, 39%, 40%, 41 % or 42%. Proanthocyanidins are molecular structures formed by oligomeric catechins and epicatechins repeats, and gallic acid esters thereof, that lead to formation of cyanidin when depolymerized under oxidative conditions. Proanthocyanidins are also called condensed tannins, as in the presence of strong acids they hydrolyze to give anthocyanidins.

The Vaccinium macrocarpon fruit extract (i) used in the present mixture (m) is preferably a water-soluble powder, more preferably a powder with an average particle size of the powder particles (or particle size distribution) lower than 250 micrometers. Preferably, this water-soluble powder may contain excipients, preferably maltodextrins, even more preferably maltodextrins made from corn starch decomposition. Preferably, a water-soluble powder : maltodextrins weight ratio ranges from 5:1 to 1 :5, more preferably from 3:1 to 1 :3.

Preferably, an aqueous solution of Vaccinium macrocarpon fruit extract (i) has a pH value ranging from 1.0 to 6.0, preferably from 2.0 to 4.5.

The mixture (m) object of the present invention comprises, in addition to extract (i), the Vaccinium myrtillus fruit extract (ii).

Vaccinium myrtillus (or bilberry) is a fruit shrub, classified as a berry-producing plant. Its fruits are small, blue pseudo-berries. They are defined as “pseudo-berries” as, despite having the appearance of berries, the ovary, sepals, petals and stamens contribute to their formation. The extract (ii) used in the mixture (m) is generated from Vaccinium myrtillus pseudo-berries.

The amount by weight of Vaccinium myrtillus fruit extract (ii) with respect to the total weight of the mixture (m) preferably ranges from 1 % to 15%, more preferably from 1 .5% to 12%, even more preferably from 2% to 9%, for example 5.2%.

The Vaccinium myrtillus fruit extract (ii) is preferably an extract titrated in anthocyanins (HPLC, method Ph. Eur. (d.e.)) in a percentage ranging from 23% to 48%, more preferably from 28% to 43%, even more preferably from 30% to 40%, further preferably from 34% to 38%, for example 34%, 35%, 36%, 37% or 38%. Anthocyanins (or anthocyans) are polyhydroxylated polyaromatic compounds capable of reacting with oxidants, such as molecular oxygen and free radicals, carrying out an antioxidant and anti-radical activity, thus reducing the damage that oxygen and radicals can cause to cells and tissues. Anthocyanins are natural phenols which, together with other families such as flavonols, flavones, isoflavones, flavanones, belong to flavonoids or bioflavonoids.

Extract (ii) is an extract obtained using an organic solvent, preferably comprising, or alternatively consisting of an, alcohol, even more preferably ethanol.

The attached Figure 2 shows a flow chart of a process for obtaining extract (ii), in accordance with a possible embodiment. In accordance with such preferred process, extract (ii) in dry form is obtained following the following steps:

(b.1 ) extraction of Vaccinium myrtillus fruits using an organic solvent, preferably an alcoholic organic solvent, even more preferably ethanol, to give an extraction solution;

(b.2) filtration of the extraction solution obtained from step (b.1 ), to give a filtered extraction solution and exhausted plant material;

(b.3) absorption of the filtered extraction solution obtained from step (b.2), to give an absorbed extraction solution;

(b.4) separation of the absorbed extraction solution obtained from step (b.3), to give a native extract and a residue / solvent;

(b.5) drying, grinding, mixing, and sieving of the native extract obtained from step (b.4), to give the dry extract (ii) and an additional residue.

Preferably, the extraction of step (b.1 ) is carried out in an extraction tank, preferably made of stainless steel.

The separation of step (b.4) is preferably carried out in a concentrator (or evaporator), more preferably a single effect one, even more preferably made of stainless steel.

By way of example, an extract (ii) usable in the present mixture (m) 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 lower than 300 pm (and therefore having a size such as to pass through a 50 Mesh sieve), more preferably with a loss on drying < 5.0%, and even more preferably with a total ash content < 5.0%.

The mixture (m) object of the present invention comprises, in addition to extract (i) and extract (ii), the Ribes nigrum fruit extract (iii). Ribes nigrum (blackcurrant or, less frequently, cassis, or Ribes nigrum L) is a plant of the Grossulariaceae family. Its fruits are in the form of globose black berries, rich in seeds, with the vestiges of the flower at their apex. Black currant differs from red currant not only in color, but also in aroma, flavor and destination of the fruits. The extract (ii) used in this mixture (m) is obtained from Ribes nigrum berries.

The amount by weight of Ribes nigrum fruit extract (iii), with respect to the total weight of mixture (m), preferably ranges from 1% to 15%, preferably from 1.5% to 12%, more preferably ranges from 2% to 9%, for example 4.6%.

Preferably, the Ribes nigrum fruit extract (iii) is an extract titrated in anthocyanins (meth. HPLC) in a percentage from 1% to 15%, preferably from 2% to 13%, more preferably from 4% to 10%, even more preferably from 6% to 8%, for example from 6%, 6.5%, 7%, 7.5% or 8%.

Anthocyanins (or anthocyanins) are compounds belonging to the flavonoid family, frequently used as water-soluble dyes. They are polyhydroxylated polyaromatic compounds capable of reacting with oxidizing agents, such as molecular oxygen or free radicals.

The extract (iii) is an extract obtained using a hydroalcoholic solvent, preferably a water-ethanol mixture. By way of example, the hydroalcoholic solvent could contain from 10% to 70% by volume of alcohol (preferably ethanol), preferably from 20% to 60% by volume, still preferably from 30% to 50% by volume, for example 40% by volume.

The attached Figure 3 shows a flow diagram of a process for obtaining extract (iii), in accordance with a possible embodiment. In accordance with such preferred process, extract (iii) in dry form is obtained following the following steps:

(c.1) Extraction of Ribes nigrum fruits with a hydroalcoholic solvent, preferably a water-ethanol mixture, to give an extraction solution;

(c.2) Optional centrifugation and/or filtration of the extraction solution obtained from step (c.1 ), to give a centrifuged and/or filtered extraction solution and exhausted plant material;

(c.3) Concentration of the extraction solution obtained from step (c.1) or the centrifuged and/or filtered extraction solution obtained from step (c.2), to give a concentrated extraction solution and a solvent; (c.4) preferably heat treatment of, and preferably addition of ingredients to the concentrated extraction solution obtained from step (c.3) to give a native extract; (c.5) Preferably homogenization-mixing and/or preferably heat treatment of the concentrated extraction solution obtained from step (c.3) or the native extract obtained from step (c.4), to give a homogenized-mixed product and/or a heat- treated product;

(c.6) Drying, preferably mixing with ingredients, and sieving the native extract obtained from step (c.4) or of the homogenized-mixed product and/or the heat- treated product obtained from step (c.5), to give the dry extract (iii) and a residue. Preferably, the extraction of step (c.1 ) is carried out in an extraction tank, more preferably made of stainless steel.

The concentration of step (c.3) is preferably carried out in a concentrator (or evaporator), more preferably a single effect one, even more preferably made of stainless steel.

The extract (iii) is preferably in a powder form, preferably a powder with a density ranging from 400 g/l to 750 g/l, more preferably from 450 g/l to 650 g/l. Even more preferably, an amount by weight ranging from 80% to 100% (preferably from 90% to 99%) of the particles of said powder has an average particle size (or average size distribution) ranging from 1 micrometer to 500 micrometers, preferably from 10 microns to 300 micron.

Preferably, the extract (iii) in a powder form has a total ash content < 5.0%, and a loss on drying < 5.0%.

Even more preferably, the extract (iii) in a powder form may contain excipients, preferably maltodextrins, even more preferably maltodextrins made from corn starch decomposition. Preferably, an [extract (iii) powder] : [maltodextrins] weight ratio ranges from 1 :10 to 10:1 , more preferably from 1 :5 to 5:1 , further preferably from 1 :3 to 3:1.

The mixture (m) object of the present invention comprises, in addition to extract (i), extract (ii) and extract (iii), the Punica granatum fruit extract (iv).

Punica granatum (or pomegranate, or Punica granatum L) is a plant belonging to the Punicaceae family, native to a geographical region extending from Iran to the Himalayan area in northern India, and which has been present since ancient times in the Caucasus and in the entire Mediterranean scrub. The fruit of the pomegranate tree is the pomegranate fruit, from which the extract (iv) contained in the mixture (m) object of the present invention is obtained.

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

The amount by weight of Punica granatum fruit extract (iv), with respect to the total weight of the above mixture (m), preferably ranges from 1 % to 15%, more preferably from 1 .5% to 11 %, even more preferably from 2% to 9%, for example 4.6%.

The attached Figure 4 shows a flow diagram of a process for obtaining extract (iv), in accordance with a possible embodiment. In accordance with such preferred process, the extract (iv) in dry form is obtained following the following steps:

(d.1 ) Supply and grinding of Punica granatum fruits, to give ground fruits;

(d.2) Maceration of the ground fruits obtained from step (d.1 ) with a hydroalcoholic solvent, preferably water-ethanol, for a time ranging from 10 minutes to 80 minutes, preferably from 20 minutes to 40 minutes, to give a macerated liquid product;

(d.3) Heating the macerated liquid obtained from step (d.2) to a temperature ranging from 50°C to 90°C, preferably from 65°C to 75°C, at atmospheric pressure, for a time ranging from 30 minutes to 4 hours, preferably from 1 hour to 2.5 hours, to give a heated liquid product;

(d.4) Centrifugation and/or filtration of the heated liquid product obtained from step (d.3), to give a centrifuged and/or filtered extraction solution and exhausted plant material;

(d.5) Concentration of the centrifuged and/or filtered extraction solution obtained from step (d.4), preferably at atmospheric pressure, more preferably at a temperature ranging from 70°C to 90°C, even more preferably from 75°C to 85°C, to give a concentrated extraction solution;

(d.6) Loading the concentrated extraction solution obtained from step (d.5) onto a chromatographic column, elution with an organic solvent, preferably an alcoholic solvent, more preferably ethanol, and collection and concentration of the eluate, to give a concentrated eluted liquid;

(d.7) Atomization of the concentrated eluted liquid obtained from step (d.6) to give an atomized product; (d.8) Pulverization of the atomized product obtained from step (d.7) to give the dry extract (iv), and preferably sieving and/or mixing said dry extract (iv).

Preferably, the maceration of step (d.2) and the heating of step (d.3) are carried out in an extraction tank, preferably made of stainless steel.

The concentration of step (b.5) is preferably carried out in a concentrator (or evaporator), more preferably a single effect one, even more preferably made of stainless steel.

Preferably, in the maceration of step (d.2), subsequent to step (d.1 ), the weight of hydroalcoholic solvent used ranges from 3 to 15 times, preferably from 5 to 10 times, even more preferably from 7 to 9 times, with respect to the weight of the ground fruit obtained from step (d.1 ).

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

The Punica granatum fruit extract (iv) is preferably an extract titrated in ellagic acid (CAS N. 476-66-4) (meth. HPLC) in a percentage ranging from 20% to 60%, preferably from 30% to 50%, more preferably from 35% to 45%, even more preferably from 38% to 42%, for example 38%, 39%, 40%, 41 % or 42%.

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

Preferably, the hydroalcoholic solvent used in step (d.2) contains from 40% to 95% by volume of alcohol (preferably ethanol), more preferably from 50% to 90% by volume, further preferably from 60% to 80% by volume, for example 70% by volume. Preferably, the extract (iv) is in a powder form. More preferably, an amount by weight ranging from 80% to 100% (preferably from 90% to 99%) of the particles of said powder has an average particle size (or average size distribution) such as to pass through an 80 mesh sieve.

Preferably, the extract (iv) in a powder form has a total ash content < 5.0%, and a loss on drying < 5.0%.

Even more preferably, the extract (iv) in a powder form may contain excipients, preferably maltodextrins, even more preferably maltodextrins made from corn starch decomposition. Preferably, an [extract (iv) powder] : [maltodextrins] weight ratio ranges from 10:1 to 1 :1 , more preferably from 8:1 to 4:1.

The mixture (m) object of the present invention comprises, in addition to extract (i), extract (ii), extract (iii) and extract (iv), the Aronia Melanocarpa fruit extract(v). Aronia Melanocarpa is a medium sized shrub, native to North America, that develops numerous, densely branched, upright stems. This shrub generates small round, pendulous fruits, that become black when ripe, and are edible. Extract (v) is obtained from said fruits.

Preferably, the amount by weight of Aronia Melanocarpa fruit extract with respect to the total weight of the above mixture (m) ranges from 15% to 50%, preferably from 20% to 45%, more preferably from 25% to 40%, for example 32.7%.

The attached Figure 5 shows a flow diagram of a process for obtaining extract (v), in accordance with a possible embodiment. In accordance with such preferred process, extract (v) in dry form is obtained as a result of a process including the following steps:

(e.1 ) Extraction of Aronia Melanocarpa fruits using a hydroalcoholic solvent, preferably a water-ethanol mixture, to give an extraction solution;

(e.2) Centrifugation and/or filtration of the extraction solution obtained from step (e.1 ), to give a centrifuged and/or filtered extraction solution (native extract) and a dry residue;

(e.3) Analysis of dry residue and polycyclic aromatic hydrocarbons (PAC)content in the native extract obtained from step (e.2);

(e.4) Optional addition of maltodextrins and/or a different batch of native extract to the native extract obtained from step (e.3);

(e.5) Concentration of the native extract obtained from step (e.3) or from step (e.4), to give a concentrated extraction solution and a solvent;

(e.6) Atomization, and preferably addition of auxiliary substances (preferably colloidal anhydrous silica), to the concentrated extraction solution obtained from step (e.5) to give a final product. Preferably, the colloidal anhydrous silica added in step (e.6) is in an amount ranging from 0.01 % to 0.5% by weight with respect to the weight of the final product.

In accordance with a possible embodiment, the extraction of step (e.1 ) is carried out in an extraction tank, preferably made of stainless steel.

In accordance with another embodiment, the concentration of step (e.5) is carried out in a concentrator (or evaporator), preferably a single-effect one, more preferably made of stainless steel.

The extract (v) is preferably in a powder form. More preferably, an amount by weight ranging from 80% to 100% (preferably from 90% to 99%) of the particles of said powder has an average particle size (or average size distribution) ranging from 1 micrometer to 700 micrometers, preferably ranging from 10 micrometers to 500 micrometers (Eur. Ph. Method, Sieve Test), even more preferably it is a powder with a loss on drying < 5.0%.

The Aronia Melanocarpa fruit extract (v) is preferably an extract titrated in proanthocyanidins (spectrophotometric method) in a percentage ranging from 0.01 % to 10%, preferably from 0.05% to 5%, more preferably from 0.1 % to 4%, even more preferably from 0.5% to 3%, for example 2%.

Preferably, the extract (v) is an extract obtained using a hydroalcoholic solvent, preferably a water-ethanol mixture. By way of example, the hydroalcoholic solvent may contain from 5% to 50% by volume of alcohol (preferably ethanol), preferably from 10% to 45% by volume, still preferably from 20% to 40% by volume, for example 30% by volume.

The mixture (m) object of the present invention comprises, in addition to extract (i), extract (ii), extract (iii), extract (iv) and extract (v), the mixture of polyphenols extracted from leaves or fruits of Olea Europaea L (vi).

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

The extract (vi) is obtained from these fruits (olives) and/or from the leaves of this tree.

Preferably, the amount by weight of the mixture of polyphenols extracted from leaves or fruits of Olea Europaea L (vi) ranges from 2% to 30%, preferably from 3% to 20%, more preferably from 5% to 18%, for example 11.8%, with respect to the total weight of the mixture (m).

Preferably, the polyphenol mixture (vi) comprises or, alternatively, consists of tyrosol, or hydroxytyrosol, or oleuropein, or mixtures thereof.

The attached Figure 6 shows a flow diagram of a process for obtaining extract (vi), in accordance with a possible embodiment. In accordance with such preferred process, the extract (vi) in dry form is obtained as a result of a process comprising the following steps:

(f.1 ) Extraction of fruits and/or leaves of Olea Europaea L using a hydroalcoholic solvent, preferably a water-ethanol mixture, to give an extraction solution;

(f.2) Centrifugation and/or filtration of the extraction solution obtained from step (f.1 ), to give a centrifuged and/or filtered extraction solution (native extract) and a dry residue;

(f.3) Analysis of dry residue and oleuropein (or tyrosol, or hydroxytyrosol) content in the native extract obtained from step (f.2);

(f.4) Optional addition of maltodextrins and/or a different batch of native extract to the native extract obtained from step (f.3);

(f.5) Concentration of the native extract obtained from step (f.3) or step (f.4), to give a concentrated extraction solution and a solvent;

(f.6) Atomization, and preferably addition of auxiliary substances (preferably colloidal anhydrous silica), to the concentrated extraction solution obtained from step (f.5) to give a final product.

Preferably, the colloidal anhydrous silica added in step (f.6) is in an amount ranging from 0.01 % to 0.5% by weight with respect to the weight of the final product.

In accordance with a possible embodiment, the extraction of step (f.1 ) is carried out in an extraction tank, preferably made of stainless steel.

In accordance with another embodiment, the concentration of step (f.5) is carried out in a concentrator (or evaporator), preferably a single effect one, more preferably made of stainless steel.

The extract (vi) is preferably in a powder form. More preferably, an amount by weight ranging from 80% to 100% (preferably from 90% to 99%) of the particles of said powder has an average particle size (or average size distribution) ranging from 1 micrometer to 700 micrometers, preferably from 10 micrometers to 500 micrometers (Eur. Ph. Method, Sieve Test), even more preferably it is a powder with a loss on drying < 5.0%.

The mixture of polyphenols extracted from leaves or fruits of Olea Europaea L (vi) is preferably an extract titrated in oleuropein (or tyrosol, or hydroxytyrosol) in a percentage ranging from 1 % to 25%, preferably from 3% to 20%, more preferably from 5% to 17%, even more preferably from 9% to 15%, for example 12%. Preferably, the extract (vi) is an extract obtained using a hydroalcoholic solvent, preferably a water-ethanol mixture. By way of example, the hydroalcoholic solvent may contain from 10% to 60% by volume of alcohol (preferably ethanol), preferably from 20% to 55% by volume, still preferably from 30% to 50% by volume, for example 40% by volume.

The mixture (m) object of the present invention comprises, in addition to extract (i), extract (ii), extract (iii), extract (iv), extract (v), and the mixture of polyphenols (vi), inulin (vii).

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

An inulin for use in the present mixture (m) may have an average length of the polymer chain ranging from 5 to 20 monomers, preferably from 6 to 15 monomers, even more preferably from 8 to 13 monomers. Preferably, inulin may be in a powder form and, more preferably, it may have an inulin content, by weight, ranging from 90% to 99%, and an overall content, by weight, of fructose, glucose and sucrose ranging from 1 % to 10%.

By way of example, inulin is inulin CAS No. 9005-80-5.

Inulin is preferably soluble inulin, that is, with a solubility in water at 25°C ranging from 5 g/l to 200 g/l.

The amount by weight of inulin (vii) with respect to the total weight of the aforementioned mixture (m) preferably ranges from 15% to 50%, preferably from 20% to 45%, more preferably from 25% to 40%, for example 36.4%.

Preferably, the mixture (m) object of the present invention comprises, in addition to extract (i), extract (ii), extract (iii), extract (iv), extract (v), the mixture of polyphenols (vi), and inulin (vii), maltodextrin, more preferably maltodextrin made from corn starch decomposition.

Preferably, a [maltodextrin] : [inulin (vii)] weight ratio ranges from 2:1 to 80:1 , more preferably from 5:1 to 50:1 , even more preferably from 10:1 to 30:1.

A further object of the present invention is a composition (c) comprising said mixture (m), and at least one physiologically and/or pharmaceutically acceptable excipient. A further object of the present invention is said composition (c) comprising said mixture (m) for use in the treatment of inflammatory diseases, both acute and chronic, preferably for use in the treatment of an inflammatory disease selected from an inflammatory bowel disease, or a systemic inflammation, preferably mild systemic inflammation.

A further object of the present invention is said composition (c) comprising said mixture (m) for use in the treatment of a condition or disorder or disease resulting from an ischemia, preferably, but not exclusively, a myocardial ischemia or ischemic/reperfusion injury to the vascular endothelium.

Preferably, the composition (c) object of the present invention is a pharmaceutical composition, or a composition for a medical device, or a composition for a dietary supplement, or a composition for a food product, or a composition for a food for special medical purposes - FSMP, said composition (c) more preferably comprising a plurality of nutrients selected from the group comprising or, alternatively, consisting of proteins, fats, vitamins, mineral elements or salts, and combinations thereof.

A further object of the present invention is a composition (c) comprising the components listed in Table 1 of Example 1.

Some examples, provided by way of non-limiting example, of the present invention will be reported below.

A mixture (m) is described herein comprising or, alternatively, consisting of (i) a Vaccinium macrocarpon fruit extract; (ii) a Vaccinium myrtillus fruit extract;

(iii) a Ribes nigrum fruit extract; (iv) a Punica granatum fruit extract; (v) an Aronia Melanocarpa fruit extract; (vi) a mixture of polyphenols extracted from leaves or fruits of Olea Europaea L; (vii) inulin. In one embodiment the mixture (m) of polyphenols extracted from leaves or fruits of Olea Europaea L (vi) comprises or, alternatively, consists of tyrosol, or hydroxytyrosol, or oleuropein, or mixtures thereof.

In one embodiment, an [extract (i)] : [extract (iii)] : [extract (iv)] weight ratio in the mixture (m) is of about 1 :1 :1.

In one embodiment, the mixture (m) comprises (in amounts expressed as percentages by weight with respect to the total weight of said mixture (m)):

(i) Vaccinium macrocarpon fruit extract ranging from 1 % to 15%, preferably from 1 .5% to 11 %, more preferably from 2% to 9%, for example 4.6%;

(ii) Vaccinium myrtillus fruit extract ranging from 1 % to 15%, preferably from 1.5% to 12%, more preferably from 2% to 9%, for example 5.2%;

(iii) Ribes nigrum fruit extract ranging from 1 % to 15%, preferably from 1 .5% to 12%, more preferably from 2% to 9%, for example 4.6%;

(iv) Punica granatum fruit extract ranging from 1 % to 15%, preferably from 1 .5% to 11 %, more preferably from 2% to 9%, for example 4.6%;

(v) Aronia Melanocarpa fruit extract ranging from 15% to 50%, preferably from 20% to 45%, more preferably from 25% to 40%, for example 32.7%;

(vi) mixture of polyphenols extracted from leaves or fruits of Olea Europaea L ranging from 2% to 30%, preferably from 3% to 20%, more preferably from 5% to 18%, for example 11 .8%;

(vii) inulin ranging from 15% to 50%, preferably from 20% to 45%, more preferably from 25% to 40%, for example 36.4%.

In one embodiment, in the mixture (m)

(i) the Vaccinium macrocarpon fruit extract is an extract titrated in proanthocyanidins at a percentage ranging from 30% to 50%, preferably from 33% to 47%, more preferably from 35% to 45%, even more preferably from 38% to 42%, for example 40%;

(ii) the Vaccinium myrtillus fruit extract is an extract titrated in anthocyanins at a percentage ranging from 23% to 48%, preferably from 28% to 43%, more preferably from 30% to 40%, even more preferably from 34% to 38%, for example 36%;

(iii) the Ribes nigrum fruit extract is an extract titrated in anthocyanins at a percentage ranging from 1 % to 15%, preferably from 2% to 13%, more preferably from 4% to 10%, even more preferably from 6% to 8%, for example 7%;

(iv) the Punica granatum fruit extract is an extract titrated in ellagic acid at a percentage ranging from 20% to 60%, preferably from 30% to 50%, more preferably from 35% to 45%, even more preferably from 38% to 42%, for example 40%;

(v) the Aronia Melanocarpa fruit extract is an extract titrated in proanthocyanidins at a percentage ranging from 0.01 % to 10%, preferably from 0.05% to 5%, more preferably from 0.1 % to 4%, even more preferably from 0.5% to 3%, for example 2%;

(vi) the mixture of polyphenols extracted from leaves or fruits of Olea Europaea L is an extract titrated in oleuropein at a percentage ranging from 1 % to 25%, preferably from 3% to 20%, more preferably from 5% to 17%, even more preferably from 9% to 15%, for example 12%;

(vii) inulin.

In one embodiment the mixture (m) further comprises maltodextrin.

In one aspect a composition (c) is provided, comprising the mixture (m) according to any one of the previous embodiments, and at least one physiologically and/or pharmaceutically acceptable excipient.

In one embodiment, the composition (c) is for use in the treatment of inflammatory diseases, preferably for use in the treatment of inflammatory diseases selected from an inflammatory bowel disease or a systemic inflammation In one embodiment, the composition (c) is for use in the treatment of a condition or a disorder or a disease resulting from an ischemia, preferably a myocardial ischemia or an alteration of the vascular endothelium function.

In one embodiment, the composition (c) is a pharmaceutical composition, or a composition for a medical device, or a composition for a dietary supplement, or a composition for a food product, or a composition for a food for special medical purposes - FSMP, said composition (c) more preferably comprising a plurality of nutrients selected from the group comprising or, alternatively, consisting of proteins, fats, vitamins, mineral elements or salts, and combinations thereof.

EXAMPLES

EXAMPLE 1

A composition (c) according to an embodiment of the present invention is shown in the following Table 1.

Table 1

Example 2

Mixture (m) according to the invention containing a combination of selected polyphenols (i) - (vi) and inulin (vii)

D.E. means dry extract

Example 3 0 1. AIM

Evaluation of the efficacy of the mixture (m) according to Example 2 in an in vitro model of intestinal epithelial cells.

MATERIALS 5 2.1 Tested Samples All extracts were diluted to a stock concentration equal to 10% of the concentration in the finished product in culture medium (Stock solution-100% solubility) and sterile filtered. The stocks were stored at -20°C.

The extracts were tested in vitro at a final concentration equal to a 1 :100 dilution in cell culture with respect to the concentration in the finished product.

2.2 Reagents and instrumentation used

3.1 Materials Cell Cultures A colon adenocarcinoma cell line, CACO-2 cells, (Foghe et al. , 1977), kept in culture in sterile flasks (25 cm ³ ), incubated at 37°C in a humid atmosphere with 5% CO2 in DMEM (Minimum Essential Medium) culture medium supplemented with 10% fetal bovine serum (FBS), 2mM glutamine, 1% FIEPES, in the presence of 1% penicillin and streptomycin, 1% L-glutamine, and 0.1% gentamycin (as in LBS n°44a protocol), is used.

The 1 :3 split is carried out every 2 days, once the monolayer is reached, by washing with 1X PBS (phosphate buffer without Ca ²⁺ and Mg ²⁺ ) and detaching the cells with a trypsin-EDTA solution at 37°C for 2 minutes.

Controls

NEGATIVE CONTROL: Untreated cells, DMEM supplemented with 10% fetal bovine serum (FBS), 2mM glutamine, 1% HEPES, in the presence of 1 % penicillin and streptomycin, 1 % L-glutamine, and 0.1 % gentamycin, and kept in 25 cm ² culture plates (12 well) at 37°C and 5% CO2.

POSITIVE CONTROL: Cells pre-treated with recombinant TNF-alpha ((7.6 ^c 10 ⁴ IU/pg) in DMEM supplemented with 10% fetal bovine serum (FBS), 2mM glutamine, 1 % FIEPES, in the presence of 1 % penicillin and streptomycin, 1 % L-glutamine, and

0.1 % gentamycin, and kept in 25 cm ² culture plates (12 well) at 37°C and 5% CO2.

3.2 Methods

3.4.1 Principle of the method

The IBS model was reproduced by pre-treating CACO-2 cells with recombinant TNF-alpha (Watari et al., 2017).

3.4.2 Experimental Procedure

Evaluation of TNF-alpha, IL-8 and IL-1beta gene expression

The cells were seeded in 12-well plates at a density equal to 10 ⁶ cells/m L. Upon reaching 80% confluence, the cells were incubated for 24h with recombinant TNF- alpha diluted to an activity of 7.6 ^c 10 ⁴ IU/pg.

At the end of the incubation, the cells were washed with PBS and incubated for 24 hours with combinations 1 to 7, respectively, as shown in the table:

The dilutions were prepared starting from stock at 10% with respect to the finished product, and diluted 1 :10 for treatment in culture medium (RPMI), supplemented with 10% fetal bovine serum (FBS), 2mM glutamine, in the presence of 1 % penicillin and streptomycin, and 0.1 % gentamycin.

In order to evaluate the effect of compounds on MICI/IBS, the gene expression of the following inflammatory markers was evaluated: TNF-alpha, IL-8 (Watari et al., 2017) and IL-l beta (Al-Sadi et al., 2010.)

The gene expression of the TNF-a marker in NCTC2544 cells was evaluated by relative quantitative RT-PCR (quantitative reverse transcription-polymerase chain reaction, qRT-PCR).

This analysis involved 3 sequential steps:

• Total RNA extraction;

• Reverse transcription into cDNA;

• qRT-PCR.

Total RNA was extracted from NCTC2544 cells according to what described by Chomczynski and Mackey (1995).

After incubation with the active compounds of interest, cells were washed with (1 *) PBS and finally subjected to an RNA extraction procedure. At the end of the extraction, the extracted RNA was quantified using the QiaExpert (Qiagen) instrument and the pg/mL concentrations of total RNA extracted were calculated at the wavelength of 260 nm.

Finally, the integrity of RNA (2 pg/mL) was assessed by an electrophoretic run on 1 % agarose gel. The total RNA was converted into cDNA (complementary DNA), using an enzyme capable of synthesizing a DNA molecule using an RNA strand as a template; this RNA-dependent DNA polymerase enzyme is called reverse transcriptase.

It binds to the 3’-end of a single RNA strand and synthesizes the cDNA strand by means of random primers and deoxynucleotide triphosphates (DNTPs).

For this purpose, a “PrimeScript™ RT Reagent Kit (perfect Real Time)” (TakaraBiolnc., Japan) commercial kit, containing 5X PrimeScript Buffer (for real Time); PrimeScript RT Enzyme Mix1 ; OligodTPrimer; Random 6 mers; RNAse free dh O, was used.

The extracted and quantified RNA was diluted to a concentration equal to 2 pg/mL and reverse transcripted into cDNA. A 10 pL Master Mix (containing 5X PrimeScript Buffer (for real Time); PrimeScript RT Enzyme Mix1 ; OligodTPrimer 50pM; Random 6 mers 100pM) was prepared, to which 10 pL of RNA (2 pg/mL) were added.

The samples were placed into a thermocycler (Stratagene Mx3000P Real Time PCR System, Agilent Technologies Italia S.p.A., Milano, Italy) and subjected to reverse transcription under the following conditions:

37°C for 15 minutes;

85°C for 5 seconds;

4°C hold.

At the end of reverse transcription, 30 pL of DEPC water were added to the samples to obtain a final cDNA concentration of 40 ng/pL. qRT-PCR is a method of amplification and real time quantification of the amplified products, by monitoring the fluorescence emitted during the reaction.

For RT-PCR amplification, the TaqMan® probe system (AppliedBiosystems) was used. The following TaqMan probes were used: Fls00174128_m1 (TNF-a), and Fls99999905_m1 (GAPDFI). GAPDFI was used as the control gene (housekeeping). The Taqman probe is a type of probe that allows the development of fluorescence while the amplification advances. A reporter (FAM™ fluorophore) is linked to its 5’- end, while a quencher is linked to its 3’-end. The proximity between the reporter and the quencher nullify the emission of the fluorescence signal. Fluorescence is detected only in the presence of 5'-exonuclease activity of the thermostable DNA polymerase (Taq polymerase) and the accumulation of the amplification products can be evaluated by the increase in the reporter fluorescence which increases during each cycle.

For qRT-PCR, a Master Mix was prepared as follows:

• 10 pL of “2* Premix Ex Taq”; · 1 pL of “20* TaqMan Gene Expression Assays” (containing 2 primers and the fluorescent probe labeled with FAM™ fluorophore);

• 0.4 pL of Rox II passive reference;

• 5 pL of DEPC water.

4 pL of cDNA for the target gene and 1 pL of cDNA for the housekeeping gene were added to the Master Mix.

The amplification was caried out under the following conditions for 40 cycles:

• 95°C, for 30 seconds (AmpliTaq activation);

• 95°C, for 5 seconds (Denaturation)

• 60°C, for 20 seconds (Annealing - extension); Each analysis was performed in duplicate.

The data obtained were analyzed according to the 2 ^_DDa method and it was thus possible to calculate the relative expression values of the gene of interest, normalized with respect to the housekeeping gene and calibrated on the control sample (untreated cells):

AACt - ACt target-housekeeping (control) -ACt target-housekeeping (treated cells)

2 - ^DDa was calculated assuming a 100% amplification efficiency. 3.4.3 Statistic Analysis

Statistical analysis was carried out using Student’s t test using Graphpad software (version 7.00 for Windows, GraphPad Software, La Jolla California USA, www.graphpad.com)

2. RESULTS

Figure 7 shows the gene expression data of TNF-alpha inflammation marker after 24 hours of incubation with the test compounds under analysis.

In the positive control (cells treated with complete culture medium and recombinant TNF-alpha) there is a significant increase in TNF-alpha.

The greatest reduction in TNF-alpha gene expression is shown in the presence of treatment with COMBINATION 1 (Compounds i-vii), and this is found to be a synergistic effect compared to the other combinations tested.

The results are shown in Figure 7 which shows a bar graph related to TNF-ct gene expression. TNF-ct gene expression in CACO-2 was evaluated by qRT-PCR. The cells were pre-treated with recombinant TNF-alpha (7.6 ^c 10 ⁴ IU/pg) for 24h. Subsequently, the cells were incubated at 37°C for 24h, 5% CO2 with:

Complete DMEM, no pre-incubation with TNF-alpha (Control);

DMEM with pre-incubation with TNF-alpha (Positive Control);

Combination 1 (Compounds i-vii);

Combination 2 (Compounds iv and v);

Combination 3 (Compounds i-vi);

Combination 4 (Compounds i-v and vii);

Combination 5 (Compounds i-v);

Combination 6 (Compound vi);

Combination 7 (Compound vii).

The values represent the Mean±SEM of two experiments carried out in duplicate.

The attached Figure 8 shows the gene expression data of the inflammation marker IL-8 after 24 hours of incubation with the compounds under analysis.

In the positive control (cells treated with complete culture medium and recombinant TNF-alpha) there is a significant reduction in IL-8.

The greatest increase in SOD-2 gene expression is shown in the presence of treatment with COMBINATION 1 (Compounds i-vii) and this is found to be a synergistic effect compared to the other combinations tested.

As shown in the attached Figure 8, IL-8 gene expression in CACO-2 cells was evaluated by qRT-PCR. The cells were pre-treated with recombinant TNF-alpha (7.6 x 10 ⁴ IU/pg) for 24h.

Subsequently the cells were incubated at 37°C for 24h, 5% CO2 with:

- Complete DMEM, no pre-incubation with TNF-alpha (Control;

- DMEM with pre-incubation with TNF-alpha (Positive Control); Combination 1 (Compounds i-vii);

Combination 2 (Compounds iv and v);

Combination 3 (Compounds i-vi);

Combination 4 (Compounds i-v and vii);

Combination 5 (Compounds i-v); Combination 6 (Compound vi);

Combination 7 (Compound vii). The values represent the Mean±SEM of two experiments carried out in duplicate.

3. BIBLIOGRAPHIC REFERENCES

Srinivasan B, Kolli AR, Esch MB, Abaci HE, Shuler ML, Hickman JJ. TEER measurement techniques for in vitro barrier model systems. J Lab Autom. 2015;20(2): 107-126. doi: 10.1177/2211068214561025

Watari A, Sakamoto Y, Hisaie K, Iwamoto K, Fueta M, Yagi K, Kondoh M: Rebeccamycin Attenuates TNF-a-lnduced Intestinal Epithelial Barrier Dysfunction by Inhibiting Myosin Light Chain Kinase Production. Cell Physiol Biochem 2017;41:1924-1934. doi: 10.1159/000472367 Al-Sadi R, Ye D, Said HM, Ma TY. IL-1 beta-induced increase in intestinal epithelial tight junction permeability is mediated by MEKK-1 activation of canonical NF-kappaB pathway. Am J Pathol. 2010;177(5):2310-2322. doi: 10.2353/ajpath.2010.100371

Chomczynski P, Mackey K. Modification of the TRI reagent procedure for isolation of RNA from polysaccharide- and proteoglycan-rich sources. Biotechniques 1995;19:942-5.

Vigetti D, Viola M, Karousou E, Rizzi M, Moretto P, Genasetti A, et al. Hyaluronan-CD44-ERK1/2 regulate human aortic smooth muscle cell motility during aging. J Biol Chem 2008;283:4448-58. La Gatta A, De Rosa M, Frezza MA, Catalano C, Meloni M, Schiraldi C.

Biophysical and biological characterization of a new line of hyaluronan-based dermal fillers: A scientific rationale to specific clinical indications. Mater Sci Eng C Mater Biol Appl. 2016;68:565-572. doi:10.1016/j.msec.2016.06.008.