Composition capable of increasing the bioavailability and absorption of molecules

Abstract

The present invention relates to a composition comprising a polysaccharide, a vegetable oil and at least one compound or molecule. Furthermore, the invention relates to the use of the composition to increase the absorption and metabolism of the at least one compound or molecule and a dietary supplement comprising said composition.

Description

[0001] DESCRIPTION

[0002] COMPOSITION CAPABLE OF INCREASING THE BIOAVAILABILITY AND ABSORPTION OF MOLECULES

[0003] The present invention relates to a composition comprising a polysaccharide, preferably pectin, at least one vegetable oil and at least one molecule or compound.

[0004] STATE OF THE ART

[0005] Bioavailability is one of the main pharmacokinetic properties and indicates the fraction of a drug that reaches systemic circulation without undergoing modifications, relative to the total amount of drug administered. By analogy with the administration of drugs, the term bioavailability is often also used with reference to food substances.

[0006] One of the mechanisms whereby the bioavailability of substances useful for the body and taken orally can be increased is to increase the intestinal absorption. Various systems can be used for this purpose, such as, for example the use of organic solvents, micronization of the substances themselves or the association of the substances with molecules having a similar biological profile.

[0007] The use of carriers of natural origin also fits into this context, as they favour the intestinal passage of the substances of interest by virtue of their intrinsic biological properties.

[0008] At present various refined vegetable oils are commonly used as carriers of natural origin to increase intestinal absorption of pharmaceutical preparations and dietary supplements.

[0009] However, such oils pose several disadvantages, prevalently correlated with the refinement process: because of this process, in fact, the oils themselves are exposed to oxidation, and the finished product contains undesirable chemical residues. Refined vegetable oils may have thus undergone a rearrangement or loss of the double bonds of the fatty acids contained in them. Consequently, the use of oils thus modified as carriers for substances to be administered orally may cause a loss of the membrane organisation of cells responsible for intestinal absorption, with a consequent loss of fluidity and permeability of the membrane itself. The rearrangement and / or loss of the double bonds may also influence the organisation and conformation of many proteins, causing various degenerative pathological conditions defined “misfolding” diseases, in particular neurodegenerative diseases (e.g. Alzheimer’s disease, Huntington’s chorea, Parkinson’s disease, amyloidosis, prion diseases), haematological diseases (for example sickle-cell anaemia), cystic fibrosis, and tumours.

[0010] In the light of the foregoing, it thus appears evident that the present use of refined vegetable oils as carriers to improve the absorption of substances useful to the body entails numerous disadvantages.

[0011] The main task of the present invention is therefore to overcome the limits of the known compositions for increasing the intestinal absorption of known substances with low bioavailability presently used according to the state of the art, in particular compositions based on refined vegetable oils. Within the scope of this task, one object of the invention is to provide a carrier composition for substances with low bioavailability which favours the absorption of such substances at the intestinal level.

[0012] Another object of the present invention is to provide a carrier composition as described above that is not harmful when administered to the body.

[0013] SUMMARY OF THE INVENTION

[0014] A first aspect of the present invention relates to a composition comprising a polysaccharide, at least one vegetable oil and at least one molecule or compound. Preferably, the at least one molecule or compound is characterised by: 1) low bioavailability; and / or 2) poor intestinal absorption; and / or 3) rapid metabolism; and / or 4) slow metabolism; and / or 5) low water solubility. Preferably, the polysaccharide is pectin.

[0015] According to one embodiment, the composition further comprises at least one vegetable oil; the at least one vegetable oil is preferably linseed oil, preferably the composition comprises a mixture of linseed oil and hemp oil.

[0016] Preferably, the volume / volume ratio between linseed oil and hemp oil ranges between 60:40 and 95:5, preferably between 65:35 and 85:15, more preferably the ratio between linseed oil and hemp oil is comprised between 70:30 and 85:15, preferably between 70:30 and 80:20; more preferably, it is 75:25.

[0017] According to one embodiment, the polysaccharide is present in a concentration of between 0.5 and 30% weight / weight (w / w) relative to the weight of the composition, preferably between 5 and 25% w / w, more preferably between 1 and 20% w / w.

[0018] Preferably, the linseed oil is present in a concentration of between 0.5 and 15 % weight / weight (w / w) relative to the weight of the composition, preferably between 1 and 10 % w / w, more preferably between 1 and 5% w / w.

[0019] Preferably, the mixture of linseed oil and hemp seed oil is present in a concentration of between 0.5 and 15% weight / weight (w / w) relative to the weight of the composition, preferably between 1 and 10% w / w, more preferably between 0.5 and 5% w / w.

[0020] Preferably, the composition comprises tryptophan.

[0021] According to one embodiment, the at least one molecule or compound is selected from: ions, preferably iron, minerals, preferably magnesium, N- acylethanolamines, preferably palmitoylethanolamide (PEA), sugars, preferably D-mannose, vitamins, preferably Vitamin D and Vitamin C, resins, preferably propolis, amino acids, preferably N-acetyl-cysteine, poly-olicarbocyclics, preferably D-chiro-lnositol, fungi and mycetes, preferably Reishi, phenols and polyphenols, preferably Gastrodia elata Blume and lycopene and curcumin, flavonoids, preferably resveratrol, isoflavonoids, bioflavonoids, phytoestrogens, preferably daidzein, carotenoids, preferably astaxanthin, medicinal herbs, plant extracts, preferably ginger and extracts thereof, Gastrodia and extracts thereof, lactic acid bacteria, lactoferrin, melatonin, coenzyme Q10 and combinations thereof.

[0022] Preferably, the composition is formulated for oral use, preferably as a lozenge, tablet, granules, pill, capsule, hard-shelled capsule, or syrup.

[0023] A second aspect of the present invention relates to the composition described above for use as a medicament.

[0024] A third aspect of the present invention relates to the composition described above for use in the prevention or treatment of a pathology selected from: a neurological, immune system, ophthalmic, urogenital, urological and gynaecological, cardiovascular, respiratory, gastrointestinal, osteoarticular, cutaneous, and muscular pathology, malabsorption syndrome and / or metabolic pathologies.

[0025] A fourth aspect of the present invention relates to the use of the composition described above as a nutraceutical, preferably as a dietary supplement, to increase the absorption or metabolism or bioavailability of compounds or molecules preferably with low bioavailability, more preferably of orally administered compounds.

[0026] A fifth aspect of the present invention relates to a dietary supplement comprising the above-described composition.

[0027] BRIEF DESCRIPTION OF THE FIGURES

[0028] Figure 1 shows an analysis of the safety of pectin alone in the formulations referred to as Method A, Method A.1 and Method B in terms of cell viability and oxidative stress at the intestinal level. The data are expressed as the mean ± SD (%) of 5 independent experiments normalised to the control;

[0029] Figure 2 shows an analysis of the product’s safety in terms of cell viability and oxidative stress with reference to Method A (A; D), Method A.1 (B; E) and Method B (C; F). The data are expressed as the mean ± SD (%) of 5 independent experiments normalised to the control. For A and D: * p<0.05 vs control; (p p<0.05 vs iron bisglycinate; # p<0.05 vs triple iron in whole pectin (Method A). For B and E: * p<0.05 vs control; (p p<0.05 vs iron bisglycinate; # p<0.05 vs triple iron in whole-fractionated pectin (Method A.1 ). Per C and F: * p<0.05 vs control; (p p<0.05 vs iron bisglycinate; # p<0.05 vs triple iron in fractionated pectin (Method B).

[0030] Figure 3 shows an analysis of the results of the absorption rate with reference to Method A (A; D), Method A.1 (B; E) and Method B (C; F). The data are expressed as the mean ± SD (%) of 5 independent experiments normalised to the control. For A and D: * p<0.05 vs control; (p p<0.05 vs iron bisglycinate; # p<0.05 vs triple iron in whole pectin (Method A). For B and E: * p<0.05 vs control; (p p<0.05 vs iron bisglycinate; # p<0.05 vs triple iron in whole-fractionated pectin (Method A.1 ). For C and F: * p<0.05 vs control; cp p<0.05 vs iron bisglycinate; # p<0.05 vs triple iron in fractionated pectin (Method B);

[0031] Figure 4 shows the functionality of the intestinal epithelium based on TEER and tight junction activity in relation to the methods referred to as Method A (A; D-F), Method A.1 (B; D-F) and Method B (C; D-F). The data are expressed as the mean ± SD (%) of 5 independent experiments normalised to the control. In the case of TEER (A; B; C): * * p<0.05 vs control; (p p<0.05 vs iron bisglycinate; # p<0.05 vs triple iron in whole pectin (Method A) for A, triple iron in whole-fractionated pectin (Method A.1 ) for B, triple iron in fractionated pectin (Method B) for C.

[0032] In the case of TJs (D; E; F): * p<0.05 vs control; (p p<0.05 vs iron bisglycinate; the bars p<0.05 vs the same Method without vitamin C;

[0033] Figure 5 shows an analysis of the product’s safety in terms of cell viability and oxidative stress with reference to Method A (A; C) and Method B (B; D). The data are expressed as the mean ± SD (%) of 5 independent experiments normalised to the control. * p<0.05 vs control; (p p<0.05 vs PEA; # p<0.05 vs PEAQ; 0 p<0.05 vs PEA linseed in whole pectin for A and C, PEA linseed in fractionated pectin for B and D;

[0034] Figure 6 shows an analysis of the results of the absorption rate with reference to Method A (A) and Method B (B). The data are expressed as the mean ± SD (%) of 5 independent experiments normalised to the control. * p<0.05 vs control; (p p<0.05 vs PEA; # p<0.05 vs PEAQ; 0 p<0.05 vs PEA linseed in whole pectin for A and PEA linseed in whole pectin for B;

[0035] Figure 7 shows an analysis of the functionality of the intestinal epithelium based on TEER and tight junction activity in relation to Method A (A; C; D; E) and Method B (B; C; D; E). The data are expressed as the mean ± SD (%) of 5 independent experiments normalised to the control. In the case of TEER (A): * p<0.05 vs control; (p p<0.05 vs PEA; # p<0.05 vs PEAQ. In the case of TEER (B):

[0036] * p<0.05 * p<0.05 vs control; (p p<0.05 vs PEA; # p<0.05 vs PEAQ; 0 p<0.05 vs PEA linseed in fractionated pectin. In the case of TJs (C, D, E): * p<0.05 vs control; the bars p<0.05 vs the same Method.

[0037] Figure 8 shows an analysis of the product’s safety in terms of cell viability and oxidative stress with reference to Method A (A; C) and Method B (B; D). The data are expressed as the mean ± SD (%) of 5 independent experiments normalised to the control. * p<0.05 vs control for A and B; (p p<0.05 vs Gastrodia; # p<0.05 vs Gastrodia Q; 0 p<0.05 vs Gastrodia linseed in whole pectin for A and C, Gastrodia linseed in fractionated pectin for B and D.

[0038] Figure 9 shows the results of the absorption rate with reference to method A (A) and method B (B). The data are expressed as the mean ± SD (%) of 5 independent experiments normalised to the control. * p<0.05 vs control; (p p<0.05 vs Gastrodia; # p<0.05 vs GastrodiaQ; 0 p<0.05 vs Gastrodia linseed in whole pectin for A and Gastrodia linseed in fractionated pectin for B.

[0039] Figure 10 shows the functionality of the intestinal epithelium based on TEER and tight junction activity in relation to Method A (A; C; D; E) and Method B (A; C; D; E). The data are expressed as the mean ± SD (%) of 5 independent experiments normalised to the control. In the case of TEER (A, B): * p<0.05 vs control; (p p<0.05 vs Gastrodia; # p<0.05 vs GastrodiaQ; 0 p<0.05 vs Gastrodia linseed in whole pectin for A and Gastrodia linseed in fractionated pectin for B. In the case of TJs (C, D, E): * p<0.05 vs control; the bars p<0.05 vs the same Method.

[0040] Figure 11 shows an analysis of the product’s safety in terms of cell viability and oxidative stress with reference to Method A (A; C) and Method B (B; D). The data are expressed as the mean ± SD (%) of 5 independent experiments normalised to the control. * p<0.05 vs control; (p p<0.05 vs mannose; # p<0.05 vs mannose Q; 0 p<0.05 vs mannose linseed in whole pectin for A, C, and mannose linseed in fractionated pectin for B, D.

[0041] Figure 12 shows an analysis of the results of the absorption rate with reference to Method A (A) and Method B (B). The data are expressed as the mean ± SD (%) of 5 independent experiments normalised to the control. * p<0.05 vs control; (p p<0.05 vs mannose; # p<0.05 vs mannose Q; 0 p<0.05 vs mannose linseed in whole pectin for A and mannose linseed in fractionated pectin for B.

[0042] Figure 13 shows an analysis of the functionality of the intestinal epithelium based on TEER and tight junction activity in relation to Method A (A; C; D; E) and Method B (A; C; D; E). The data are expressed as the mean ± SD (%) of 5 independent experiments normalised to the control. In the case of TEER (A, B): * p<0.05 vs control; (p p<0.05 vs mannose; # p<0.05 vs mannose Q; 0 p<0.05 vs mannose linseed in whole pectin for A and mannose linseed in fractionated pectin for B. In the case of TJs (C, D, E): * p<0.05 vs control; the bars p<0.05 vs the same Method.

[0043] Figure 14 shows an analysis of the product’s safety in terms of cell viability and oxidative stress with reference to Method A (A; C) and Method B (B; D). The data are expressed as the mean ± SD (%) of 5 independent experiments normalised to the control. * p<0.05 vs control; (p p<0.05 vs ginger; # p<0.05 vs ginger Q; 0 p<0.05 vs ginger linseed in whole pectin for A and ginger linseed in fractionated pectin for B.

[0044] Figure 15 shows an analysis of the results of the absorption rate with reference to Method A (A) and Method B (B). The data are expressed as the mean ± SD (%) of 5 independent experiments normalised to the control. * p<0.05 vs control; (p p<0.05 vs ginger; # p<0.05 ginger Q; 0 p<0.05 vs ginger linseed in whole pectin for A and ginger linseed in fractionated pectin for B.

[0045] Figure 16 shows an analysis of the functionality of the intestinal epithelium based on TEER and tight junction activity in relation to Method A (A; C; D; E) and Method B (A; C; D; E). The data are expressed as the mean ± SD (%) of 5 independent experiments normalised to the control. In the case of TEER (A, B): * p<0.05 vs control; (p p<0.05 vs ginger; # p<0.05 vs ginger Q; 0 p<0.05 vs ginger linseed in whole pectin for A and ginger linseed in fractionated pectin for B. In the case of TJs (C, D, E): * p<0.05 vs control; the bars p<0.05 vs the same Method.

[0046] Figure 17 shows an analysis of the functionality of the intestinal epithelium based on TEER (A) and tight junction activity (B) in relation to the composition comprising DCI and pectin obtained from citrus fruit (Dyno 1) or pectin obtained from apple (Dyno 2), dissolved hot or cold in the solvent. The data are expressed as the mean ± SD (%) of 5 independent experiments conducted in triplicate normalised to the control. * p<0.05 vs Control; a p<0.05 vs DCI; p p<0.05 vs DCIQ; y p<0.05 vs the other forms of Dyno.

[0047] Figure 18 shows an analysis of intestinal absorption in relation to a composition comprising DCI and Dyno 1 or Dyno 2, dissolved hot or cold in the solvent. A: the data are expressed as the mean ± SD (%) of 5 independent experiments conducted in triplicate normalised to the control. B: the values derive from the application of the formula [doi: 10.3390 / brainsci10070457]: J = Jmax [C] / (Kt + [C]);C: initial concentration of fluorescein; Jmax: the maximum permeation rate; Kt: the Michaelis-Menten constant.

[0048] Figure 19 shows an analysis of intestinal epithelium functionality based on TEER (A) and tight junction activity (B) in relation to the composition comprising KCI and Dyno 1 pectin or Dyno 2 pectin, dissolved hot or cold in the solvent. The data are expressed as the mean ± SD (%) of 5 independent experiments normalised to the control. * p<0.05 vs Control; a p<0.05 vs KCI; p<0.05 vs KCIQ; y p<0.05 vs the other forms of Dyno. Figure 20 shows an analysis of intestinal absorption in relation to the composition comprising KCI and Dyno 1 or Dyno 2, dissolved hot or cold in the solvent. A: values of Papp < 0.2 x 10-6 cm / s indicate a low absorption with a bioavailability < 1%, values between 0.2 x 10-6 and 2 x 10-6 cm / s indicate a bioavailability of between 1 and 90%, and values > 2 x 10-6 cm / s indicate excellent absorption, with a bioavailability greater than 90%. B: the values derive from the application of the formula [doi: 10.3390 / brainsci10070457]: J = Jmax [C] / (Kt + [C]);C: initial concentration of fluorescein; Jmax: the maximum permeation rate; Kt: the Michaelis-Menten constant.

[0049] Figure 21 shows an analysis of intestinal absorption in relation to the composition comprising mulberry, Dyno 1 pectin or Dyno 2 pectin, dissolved hot or cold in the solvent, and tryptophan. The reported data are expressed as the mean ± SD of 5 independent experiments conducted in triplicate. All the substances p<0.05 vs the control; a p<0.05 vs mulberry leaf extract; p p<0.05 vs mulberry leaf extract Dyno 2; y p<0.05 vs mulberry leaf extract DynoQ 2; 5 p<0.05 vs mulberry leaf extract Dyno 1 .

[0050] Figure 22 shows an analysis of the production of insulin and its receptor substrates (IRS1) in relation to the composition comprising mulberry, Dyno 1 pectin or Dyno 2 pectin, dissolved hot or cold in the solvent, and tryptophan. The reported data are expressed as the mean ± SD of 5 independent experiments conducted in triplicate. * p<0.05 vs control, a p<0.05 vs mulberry leaf extract; p<0.05 vs mulberry leaf extract Dyno 2; y p<0.05 vs mulberry leaf extract DynoQ 2; 5 p<0.05 vs mulberry leaf extract Dyno 1 .

[0051] Figure 23 shows an analysis of the reduction in circulating glucose in relation to the composition comprising mulberry, Dyno 1 pectin or Dyno 2 pectin, dissolved hot or cold in the solvent, and tryptophan. The reported data are expressed as the mean ± SD of 5 independent experiments conducted in triplicate. * p<0.05 vs control, a p<0.05 vs mulberry leaf extract; p p<0.05 vs mulberry leaf extract Dyno 2; y p<0.05 vs mulberry leaf extract DynoQ 2; 5 p<0.05 vs mulberry leaf extract Dyno 1 .

[0052] Figure 24 shows an analysis of the activity of DMT-1 and ferritin to assess the effects of triple iron in the composition with Dyno 1 pectin or Dyno 2 pectin, dissolved hot or cold in the solvent, and tryptophan. The reported data are expressed as the mean ± SD of 5 independent experiments conducted in triplicate. * p<0.05 vs control, cpp<0.05 vs all the other samples.

[0053] DEFINITIONS

[0054] In the context of the present invention, the term “pectin” means a natural polymer, consisting of galacturonic acid units linked by a(1-4) bonds in variably branched chains. In a variable percentage, the carboxylic groups of the polymer are esterified with CH3OH (methanol); this percentage defines its degree of methoxylation (DM).

[0055] In the context of the present invention, linseed oil, or more simply “flax oil”, means the oil obtained by pressing the ripened seeds of the flax plant (Li num usitatissimum L.), which have usually been previously dried. Linseed oil is mainly composed of triglycerides and, generally, the typical distribution of the fatty acids making it up is the following: about 10% saturated fatty acids, about 23% monounsaturated fatty acids, and 67% polyunsaturated fatty acids. Among the polyunsaturated fatty acids, linseed oil is rich mainly in linoleic acid (omega-6) and a-linolenic acid (omega-3), in a ratio of about 1 :4.

[0056] In the context of the present invention, hemp seed oil, or simply “hemp oil”, means a product extracted from the fruits of Cannabis sativa L., generally by cold pressing.

[0057] Cold-pressed hemp seed oil contains about 10% saturated fatty acids and about 90% unsaturated fatty acids, in particular, about 50-60% are omega-6 and about 20% are omega-3.

[0058] Hemp seed oil is thus the vegetable oil with the highest content of unsaturated fatty acids. Furthermore, hemp oil has a high content of vitamin E (whose antioxidant action is known, carotene (precursor of vitamin A), phytosterols, phospholipids and minerals, including calcium, magnesium, sulphur, potassium, and phosphorous, as well as modest amounts of iron and zinc.

[0059] DETAILED DESCRIPTION OF THE INVENTION

[0060] A first aspect of the present invention relates to a composition comprising a polysaccharide, preferably a heteropolysaccharide, and at least one compound or molecule. According to a preferred embodiment of the invention, the polysaccharide is pectin. Preferably, the polysaccharide, more preferably the pectin, is of natural origin, obtained from citrus peel or apple.

[0061] In one embodiment, the pectin is selected from high methoxyl pectins and low methoxyl pectins.

[0062] High methoxyl pectins gel optimally in the absence of bivalent calcium ions and under acidic pH conditions (2-3.8), with a high concentration of soluble solids (SS > 65%), typically generating non-heat-reversible gels. Low methoxyl pectins, by contrast, gel in the presence of bivalent calcium ions and in a wider pH and SS range - respectively 2.6-7 and 10-70%.

[0063] According to a preferred embodiment of the invention, the pectin is low methoxyl pectin.

[0064] A particular variety of low methoxyl pectins defined as amidated pectins have the advantage, compared to conventional pectins, of showing a gelation dynamic that is more unconstrained by the presence of calcium ions. Amidation further renders the pectin able to give rise to heat- reversible gels, whereas conventional low methoxyl pectins give rise to heat-resistant gels.

[0065] According to a preferred embodiment of the invention, the pectin is an amidated pectin, preferably an amidated low methoxyl pectin.

[0066] Preferably, the pectin was dissolved at a temperature of between 35 and 40 °C in a solvent, preferably in water, before the addition of the other components.

[0067] Or else, the pectin was dissolved at a temperature of between 15 and 25 °C in a solvent, preferably in water, before the addition of the other components.

[0068] According to a further embodiment, the pectin is derived / obtained from at least one fruit; the pectin is preferably obtained from apple and / or a citrus fruit.

[0069] According to one embodiment, the composition further comprises at least one vegetable oil, preferably at least two vegetable oils. Preferably, the at least one oil is linseed oil and / or hemp seed oil.

[0070] According to a preferred embodiment of the invention, the composition comprises pectin and linseed oil.

[0071] In another embodiment, the composition comprises pectin and a mixture of linseed oil and hemp oil.

[0072] According to one embodiment of the invention, the composition comprises the mixture of linseed oil and hemp oil in a linseed oikhemp oil volume / volume ratio of between 60:40 and 95:5, preferably between 65:35 and 85:15. According to a further preferred embodiment, the composition comprises the mixture of linseed oil and hemp oil in a linseed oikhemp oil ratio of between 70:30 and 85:15; more preferably, the composition comprises the mixture of linseed oil and hemp oil in a linseed oikhemp oil ratio of between 70:30 and 80:20.

[0073] In a particularly preferred embodiment of the invention, the ratio between linseed oil and hemp oil is 75:25.

[0074] In the present text, where reference is made to the ratio between linseed oil and hemp oil, it is understood that the ratio is expressed as volume / volume, for example a volume / volume ratio comprised between 60:40 means that, out of a volume of 100 ml, 60 ml are linseed oil and 40 ml are hemp seed oik An analogous reasoning applies in reference to different ratios.

[0075] In any case, in this context, the ratio between linseed oil and hemp oil can be modified within the range envisaged by the present invention, in particular with the aim of optimising the effectiveness of the composition, for example based on one or more further substances that may be present in the composition itself, as described below.

[0076] According to a preferred aspect of the present invention, the linseed oil and / or hemp oil are unrefined oils.

[0077] Preferably, the linseed oil and / or hemp oil are obtained by cold pressing.

[0078] According to one embodiment, the composition comprises a percentage of polysaccharide of between 0.5 and 30% weight / weight (w / w) relative to the weight of the composition, preferably between 5 and 25% w / w, more preferably between 10 and 20 % w / w.

[0079] Preferably, the composition comprises a percentage of linseed oil of between 0.5 and 15% weight / weight (w / w) relative to the weight of the composition, preferably between 1 and 10% w / w, more preferably between 1 and 5% w / w.

[0080] Preferably, the composition comprises the mixture of linseed oil and hemp seed oil described above in a percentage of between 0.5 and 15% weight / weight (w / w) relative to the weight of the composition, preferably between 1 and 10% w / w, more preferably between and 1 and 5% w / w.

[0081] According to one embodiment, the composition further comprises at least one amino acid that is essential for the human body, preferably tryptophan.

[0082] Preferably, the composition comprises tryptophan in a concentration of between 0.05 and 2% weight / weight of the composition, preferably between 0.8 and 1% more preferably between 0.1 and 0.5 % w / w.

[0083] Therefore, in one embodiment, the composition comprises pectin, a vegetable oil, tryptophan and at least one molecule or compound, as defined herein.

[0084] Advantageously, the above-described composition is characterised by high plasma absorption or an improvement of the absorption kinetics (pharmacodynamics), that is, it shows to be superior compared to the absorption of the compositions as such or known lipid compositions presently employed. This ability to be easily absorbed at the intestinal level by entering into the bloodstream enables the composition of the present invention to represent an effective system for allowing the entry into plasma also of other substances with low or reduced bioavailability, or rapid metabolism. In other words, the composition described is a carrier, a system for delivering, in an optimal manner, substances and compounds, preferably those characterised by a low or reduced bioavailability, poor intestinal absorption or rapid metabolism.

[0085] In fact, many of the substances that are useful or in any case beneficial for the human body have low bioavailability, often due to poor absorption at the intestinal level.

[0086] Therefore, the composition of the invention is particularly useful for the purpose of facilitating intestinal absorption of substances (molecules / compounds) commonly employed in the nutraceutical and / or pharmaceutical sector, preferably natural extracts of plant and / or animal origin with low bioavailability and / or poor absorption, which can be more or less lipophilic.

[0087] According to a preferred embodiment of the invention, the composition further comprises at least one compound or molecule. Preferably, said at least one compound or molecule are substances that are commonly employed in the nutraceutical or pharmaceutical and preferably have low bioavailability due to their reduced intestinal absorption.

[0088] According to one embodiment, the composition comprises at least one molecule or compound characterised by: 1) low bioavailability; and / or 2) poor intestinal absorption; and / or 3) rapid metabolism; and / or 4) slow metabolism; and / or 5) low water solubility, more preferably said molecule or compound being selected from: ions, preferably iron, minerals, preferably magnesium, N- acylethanolamines, potassium chloride, preferably palmitoylethanolamide (PEA), sugars, preferably D-mannose, vitamins preferably Vitamin D and Vitamin C, resins, preferably propolis, amino acids, preferably N-acetyl-cysteine, poly- oligocarbocyclics, preferably D-chiro-lnositol, fungi and mycetes, preferably Reishi, phenols and polyphenols, preferably Gastrodia elata Blume and lycopene and curcumin, flavonoids, preferably resveratrol, isoflavonoids, bioflavonoids, phytoestrogens, preferably daidzein, carotenoids, preferably astaxanthin, medicinal herbs, plant extracts, preferably ginger and extracts thereof, Gastrodia and extracts thereof, lactic acid bacteria, lactoferrin, melatonin, coenzyme Q10, mulberry extract and combinations thereof.

[0089] In a preferred embodiment, the composition further comprises at least one compound selected from: iron, PEA, Gastrodia, D-mannose, D-chiro-lnositol, potassium chloride, propolis, ginger and vitamin C, mulberry extract and combinations thereof.

[0090] According to one embodiment, the composition comprising the pectin and linseed oil in the ratios described above further comprises at least compound selected from: iron, PEA, Gastrodia, D-mannose, D-chiro-lnositol, propolis, ginger and vitamin C and combinations thereof.

[0091] According to a particularly preferred embodiment the composition comprising the pectin and the mixture of linseed oil and the hemp oil in the ratios described above further comprises at least one compound selected from: iron, PEA, Gastrodia, D-mannose, D-chiro-lnositol, propolis, ginger and vitamin C and combinations thereof.

[0092] According to a further preferred embodiment of the invention, the poorly bioavailable substances are also molecules whose metabolism is particularly rapid, and / or molecules having low water solubility, that is, lipophilic substances / molecules.

[0093] Preferably, the composition comprises at least one compound or molecule in a percentage of between 1 and 85% weight / weight (w / w) relative to the weight of the composition, preferably between 5 and 80% w / w, more preferably between 10 and 60% w / w.

[0094] Preferably, the composition comprises PEA in a percentage of between 15 and 75% weight / weight (w / w) relative to the weight of the composition, preferably between 20 and 70% w / w, more preferably between 30 and 60% w / w.

[0095] Preferably, the composition comprises D-chiro-lnositol in a percentage of between 5 and 35% weight / weight (w / w) relative to the weight of the composition, preferably between 8 and 30% w / w, more preferably between 10 and 20% w / w.

[0096] Preferably, the composition comprises Gastrodia in a percentage of between 5 and 75% weight / weight (w / w) relative to the weight of the composition, preferably between 8 and 70% w / w, more preferably between 10 and 60% w / w.

[0097] Preferably, the composition comprises ginger in a percentage of between 5 and 75% weight / weight (w / w) relative to the weight of the composition, preferably between 8 and 70% w / w, more preferably between 10 and 60% w / w.

[0098] Preferably, the composition comprises mannose in a percentage of between 15 and 85 % weight / weight (w / w) relative to the weight of the composition, preferably between 20 and 80% w / w, more preferably between 30 and 70% w / w.

[0099] In one embodiment, the composition further comprises at least one excipient and / or preservative and / or pH stabiliser suitable for pharmaceutical and / or nutraceutical use.

[0100] According to one embodiment, the composition described above in detail is formulated for oral use, preferably as a lozenge, tablet, granules, pill, capsule, hard-shelled capsule, or syrup.

[0101] According to a preferred embodiment, the composition of the invention is prepared in a conventional manner, by adding the at least one compound or molecule to the linseed oil and subsequently mixing with the polysaccharide, preferably pectin.

[0102] Preferably, the composition is obtained by adding the at least one compound or molecule to the mixture of linseed oil and hemp oil and subsequently mixing with the polysaccharide, preferably pectin.

[0103] In the case of preparations with several ingredients, the latter can also be dissolved in only one of the two oils separately and be mixed together only at the end.

[0104] In other words, the at least one compound or molecule and the at least one vegetable oil, preferably linseed oil, more preferably the mixture of oils described above, are incorporated into the polysaccharide, preferably pectin, which performs an excipient function.

[0105] A second aspect of the present invention relates to the composition described above in detail for use as a medicament.

[0106] A third aspect of the present invention relates to the medical use of the composition as described above, in particular for prevention and / or therapeutic treatment and / or during the follow-up phase or later (to reduce the risk of relapse) of a pathology selected from: a neurological, immune system, ophthalmic, urogenital, urological and gynaecological, cardiovascular, respiratory, gastrointestinal, osteoarticular, cutaneous, and muscular pathology, malabsorption syndrome and / or metabolic pathologies.

[0107] A fourth aspect of the present invention relates to the use of the composition described above in detail as a nutraceutical, food for special medical purposes, functional food or dietary supplement.

[0108] In general, the composition as described above is used to increase the absorption and / or metabolism and / or bioavailability, preferably at the intestinal level, of compounds or molecules, preferably with low bioavailability or poor intestinal absorption, more preferably of orally administered compounds. Preferably, the composition is used to increase the absorption and / or metabolism and / or bioavailability and / or absorption kinetics, preferably at the intestinal level, of at least one compound or molecule selected from: ions, preferably iron, minerals, preferably magnesium, N-acylethanolamines, preferably palmitoylethanolamide (PEA), sugars, preferably D-mannose, vitamins preferably Vitamin D and Vitamin C, resins, preferably propolis, amino acids, preferably N- acetyl-cysteine, poly-olicarbocyclics, preferably D-chiro-lnositol, fungi and mycetes, preferably Reishi, phenols and polyphenols, preferably Gastrodia elata Blume and lycopene and curcumin, flavonoids, preferably resveratrol, isoflavonoids, bioflavonoids, phytoestrogens, preferably daidzein, carotenoids, preferably astaxanthin, medicinal herbs, plant extracts, preferably ginger and extracts thereof, Gastrodia and extracts thereof, lactic acid bacteria, lactoferrin, melatonin, coenzyme Q10 and combinations thereof.

[0109] A fifth aspect of the present invention relates to a dietary supplement comprising the composition described above in detail.

[0110] A sixth aspect of the present invention relates to a method for increasing the bioavailability of a molecule or compound in an individual. Said method comprises at least one step of administering the composition described above in detail to an individual who has a need therefor. According to one embodiment, said individual is a human individual or animal.

[0111] EXAMPLE

[0112] Example 1 Cell cultures

[0113] The human intestinal epithelial cell line (Caco-2) was used as an experimental model to predict the characteristics of intestinal absorption following oral intake [DiMarco, R.L.; Hunt, D.R.; Dewi, R.E.; Heilshorn, S.C. Improvement of paracellular transport in the Caco-2 drug screening model using protein- engineered substrates. Biomaterials 2017, 129, 152-162], This cell line was cultured in Advanced Dulbecco's Modified Eagle's Medium / Nutrient F-12 Ham (Adv DMEM-F12; GIBCO® ThermoFisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (FBS, Merck Life Science, Rome, Italy), 2 mM L-glutamine and 1% penicillin-streptomycin (Merck Life Science, Rome, Italy) and kept in an incubator at 37°C with 5% CO2[Galla, R.; Ruga, S.; Aprile, S.; Ferrari, S.; Brovero, A.; Grosa, G.; Molinari, C.; Uberti, F. New Hyaluronic Acid from Plant Origin to Improve Joint Protection-An In Vitro Study. Int. J. Mol. Sci. 2022, 23, 8114.]. For the purpose of conducting the experiments, Caco-2 cells were used with a passage number of between 26 and 32 to maintain the correct properties of permeability and paracellular transport, similar to the mechanism of intestinal absorption following oral intake in humans [Uberti, F.; Morsanuto, V.; Ruga, S.; Galla, R.; Farghali, M.; Notte, F.; Bozzo, C.; Magnani, C.; Nardone, A.; Molinari, C. Study of Magnesium Formulations on Intestinal Cells to Influence Myometrium Cell Relaxation. Nutrients 2020, 12, 573].

[0114] MTT cell viability assay

[0115] After treatment, the analysis of cell viability was conducted with a classic technique based on the MTT in vitro toxicology assay kit (Merck Life Science, Rome, Italy) [Ruga, S.; Galla, R.; Penna, C.; Molinari, C.; Uberti, F. The Activity of Ten Natural Extracts Combined in a Unique Blend to Maintain Cholesterol Homeostasis-In Vitro Model. Int. J. Mol. Sci. 2022, 23, 3805.], following the manufacturer’s instructions. In fact, at the end of the treatment, the cells were incubated with 1% MTT dye for 2 hours in an incubator at 37 °C, with 5% CO2and 95% humidity; then the purple formazan crystals were dissolved in an equal volume of MTT solubilisation solution. Absorbance was analysed by means of a spectrophotometer (Infinite 200 Pro MPlex, Tecan, Mannedorf, Switzerland) at 570 nm with correction at 690 nm, and the results were expressed versus the control (0% line), which represented the untreated cells. The results represent the percentage of viable cells versus the control, which makes it possible to understand whether the stimulation is safe or not.

[0116] Production of reactive oxygen species (ROS)

[0117] Quantification of the release of superoxide anions was obtained following a standard protocol based on the reduction of cytochrome c [Molinari C, Ruga S, Farghali M, Galla R, Fernandez-Godino R, Clemente N, Uberti F. Effects of a New Combination of Natural Extracts on Glaucoma-Related Retinal Degeneration. Foods. 2021 Aug 15;10(8) :1885. doi: 10.3390 / foods10081885. PMID: 34441662; PMCID: PMC8391439.]. Absorbance was measured in the culture supernatants at 550 nm using a spectrophotometer (Infinite 200 Pro MPlex, Tecan, Mannedorf, Switzerland). In particular, 100 pL of cytochrome c (Merck, Milan, Italy) were added to all the wells, whilst 100 pL of superoxide dismutase (Merck, Milan, Italy) and 100 pL of cytochrome c were added to the empty wells; the plate was then incubated for 30 minutes. Subsequently, 100 pL were drawn from each well and the absorbance was measured with a spectrophotometer (Infinite 200 Pro MPlex, Tecan, Mannedorf, Switzerland) at 550 nm. The O2rate was expressed as the mean ± SD (%) of nanomoles of reduced cytochrome c per microgram of protein versus the control (0 line).

[0118] Experimental model of intestinal barrier

[0119] A model of the intestinal barrier was constructed using Caco-2 cells in order to analyse the passage of the substances under examination through the intestinal barrier. For this reason, the TEER values were determined with EVOM3, coupled with STX2 chopstick electrodes (World Precision Instruments, Sarasota, FL, USA); this assay was carried out every 2 days for 21 days until reaching a TEER value > 400 Qcm2 before the stimulation [Zhao, X., Xu, X. X., Liu, Y., Xi, E. Z., An, J. J., Tabys, D., & Liu, N. (2019). The In Vitro Protective Role of Bovine Lactoferrin on Intestinal Epithelial Barrier. Molecules (Basel, Switzerland), 24(1), 148]; this time is necessary for the formation of a cell monolayer, cell differentiation and exposure of the intestinal villi. On day 21 , the culture medium at the apical and basolateral levels was changed to create different pH conditions: pH of around 6.5 at the apical level (acidic pH that imitates the lumen of the small intestine) and pH of around 7.4 at the basolateral level (neutral pH that imitates human blood) [Uberti, F., Morsanuto, V., Ghirlanda, S., & Molinari, C. (2017). Iron Absorption from Three Commercially Available Supplements in Gastrointestinal Cell Lines. Nutrients, 9(9), 1008. https: / / doi.org / 10.3390 / nu9091008]. The cells were maintained for 15 minutes at 37 °C with 5% CO2, after which the TEER values were measured again before the beginning of the experiment to verify the stabilisation of the values. At the end of the stimulation interval, the medium of the basolateral environment was collected so that it could be used to stimulate the peripheral nerve model.

[0120] Analysis of intestinal permeability

[0121] The measurement of TEER is used to assess the barrier function of epithelial cells on these porous supports. This test was carried out every 2 days for 21 days until reaching a TEER value > 400 Qcm2 before the stimulation. Once the threshold value was reached, the fluorescent probe, 0.04% fluorescein (Merck Life Science, Rome, Italy), was added together with the treatments and determined in the apical and basolateral compartments by converting the amount of total volume in relation to the surface area of the Transwell (pg / cm2), according to a classic method. Briefly, the values of absorption through intestinal epithelial cells derive from the application of the following formula [Morsanuto, V., Galla, R., Molinari, C., & Uberti, F. (2020). A New Palmitoylethanolamide Form Combined with Antioxidant Molecules to Improve Its Effectiveness on Neuronal Aging. Brain sciences, 10(7), 457]:

[0122] J = Jmax [C] / (Kt + [C])

[0123] C: initial concentration of fluorescein

[0124] Jmax: the maximum permeation rate

[0125] Kt: the Michaelis-Menten constant

[0126] The negative controls without cells were analysed to rule out the influence of the Transwell membrane. Fluorescence was detected with a fluorescence spectrophotometer (Infinite 200 Pro MPlex plate reader, Tecan, Mannedorf, Switzerland) at excitation / emission wavelengths of 490 / 514 nm. The results are expressed as the proportion of the original amount that permeated through the cells.

[0127] Analysis of Claudin-1

[0128] Human Claudin 1 was measured in the Caco-2 lysates by means of an ELISA kit (Cusabio Technology LLC, Houston, TX, USA), following the manufacturer’s instructions. Briefly, the cells were lysed with cold PBS 1 x (Merck Life Science, Rome, Italy) and centrifuged at 1500x g for 10 minutes at 4 °C. Then 100 pL of each sample were added to the ELISA plate and incubated at 37 °C for 2 hours; afterwards, the plate was washed and 100 pL of biotin antibody were added to the wells and incubated for 1 hour at 37 °C. After this time, the wells were washed and 100 pL of HRP-avidin were added to each well; the samples were incubated for 1 h at 37 °C. Subsequently, 90 pL of TMB substrate were also added to the samples and the plate was incubated for 20 minutes at 37 °C, away from light. At the end, 50 pL of stop solution were used to halt the reaction and the plate was analysed with a spectrophotometer (Infinite 200 Pro MPlex, Tecan, Mannedorf, Switzerland) at 450 nm. The concentration was expressed as pg / mL, comparing the data with the standard curve (range from 0 to 1000 pg / mL) and the results were expressed as a percentage (%) versus the control (0 line) of five independent experiments carried out in triplicate [R. Galla, P. Grisenti, M. Farghali, et aL, Ovotransferrin Supplementation Improves the Iron Absorption: An In Vitro Gastro-Intestinal Model. Biomedicines, 9(11 ) (2021 ) 1543].

[0129] Analysis of Occludin

[0130] The ELISA kit for human Occludin (OCLN kit, MyBiosource, San Diego, CA, USA) was used to analyse the presence of Occludin in the Caco-2 cell lysates, according to the manufacturer’s instructions. Briefly, the Caco-2 cells were lysed with PBS 1 x (Merck Life Science, Rome, Italy), centrifuged at 1500x g for 10 minutes at 4 °C and 100 pL of each sample were transferred into the specific well prior to incubation at 37 °C for 90 minutes. The supernatants were removed, and the plate was incubated with 100 pL of detection solution A for 45 minutes at 37 °C; the wells were then washed with the washing solution and incubated with 100 pL of detection solution B for another 45 minutes. At the end of this time, 90 pL of substrate solution were added, followed by 20 minutes’ incubation at 37 °C in darkness; then 50 pL of stop solution were used to block the enzymatic reaction. The plate was analysed with a spectrophotometer (Infinite 200 Pro MPlex, Tecan, Mannedorf, Switzerland) at a wavelength of 450 nm. The concentration is expressed in pg / mL, compared to a standard curve (range from 0 to 1500 pg / mL) and the results are expressed in percentage (%) versus the control (0 line) of five independent experiments carried out in triplicate [R. Galla, S. Ruga, S. Aprile, et aL, New Hyaluronic Acid from Plant Origin to Improve Joint Protection-An In Vitro Study. Int. J. Mol. Sci., 23(15) (2022) 8114],

[0131] Analysis of ZO-1 The Human Tight Junction Protein 1 ELISA kit (MyBiosource, San Diego, CA, USA) was used for measurement in the Caco-2 cells, following the manufacturer’s instructions. Briefly, the cells were washed with cold phosphate- buffered saline 1 x (PBS 1 x, Merck Life Science, Rome, Italy) and processed with two cycles of freezing / thawing; then the cell lysates were centrifuged for 5 minutes at 5000xg at 4 °C. Afterwards, 100 pL of each sample were collected and incubated on the ELISA plate at 37 °C for 90 minutes; after washing, 100 pL of detection solution A were added to each well, followed by 45 minutes’ incubation at 37 °C. The wells were washed and 100 pL of detection solution B were added to the samples. After 45 minutes’ incubation, the wells were washed again and 90 pL of substrate solution were added to each well; the samples were then incubated for 20 minutes at 37 °C in darkness. Finally, 50 pL of stop solution were added and the plates were read with a spectrophotometer (Infinite 200 Pro MPlex, Tecan, Mannedorf, Switzerland) at a wavelength of 450 nm. The concentration was expressed as pg / mL, comparing the data with the standard curve (range from 0 to 1000 pg / mL), and the results were expressed as a percentage (%) versus the control (0 line). [R. Galla, S. Ruga, S. Aprile, et aL, New Hyaluronic Acid from Plant Origin to Improve Joint Protection-An In Vitro Study. Int. J. Mol. Sci., 23(15) (2022) 8114], Experimental protocol

[0132] For the development of this project, the Caco-2 cells were seeded differently in order to conduct different experiments, including 1 x 104cells in 96-well plates to investigate cell viability using the MTT toxicology assay (Merck Life Science). Eight hours before stimulation, the cells were incubated with Adv DMEM without phenol red and FBS (GIBCO® ThermoFisher Scientific, Waltham, MA, USA), 2 mM of L-glutamine and 1% penicillin-streptomycin (both from Merck Life Science, Rome, Italy) at 37 °C in order to synchronise them. Furthermore, 2 x 104cells were placed in a 6.5 mm Transwell® (Corning® Costar®, Merck Life Science, Rome, Italy) with an insert consisting of a polycarbonate membrane with 0.4 pm pores (Corning® Costar®, Merck Life Science, Rome, Italy), in a 24-well plate in order to carry out the absorption analysis. The cells seeded into the Transwell® insert were maintained in a complete culture medium, changed every other day on the basal and apical sides for 21 days before the stimulations [Uberti, F.; Morsanuto, V.; Ghirlanda, S.; Molinari, C. Iron Absorption from Three Commercially Available Supplements in Gastrointestinal Cell Lines. Nutrients 2017, 9, 1008]. Before the stimulation, on the apical side, the culture medium was brought to a pH of 6.5 with the aim of mimicking the pH of the lumen of the small intestine, whereas at the basolateral level the pH was maintained at around 7.4, which represents blood [Hubatsch, I.; Ragnarsson, E.G.; Artursson, P. Determination of drug permeability and prediction of drug absorption in Caco-2 monolayers. Nat. Protoc. 2007, 2, 2111-2119.]. This in vitro model has been validated by the EMA and FDA for predicting the absorption, metabolism and bioavailability of drugs, metabolism and bioavailability of various substances after oral administration in humans [Fda.Gov. Available online: https: / / www.fda.gov / mediaZ117974 / download (accessed on 2 November 2023)]. Sample preparation 1

[0133] METHOD A. The samples under examination were tested at the level of intestinal cells: in individual form dissolved in a culture medium and in a preferred mixture of linseed and hemp oil or in linseed oil alone; in dissolved form in the medium and a preferred mixture of linseed and hemp oil or in linseed oil alone, but incorporated into pectin, so as to form small tablets. Specifically, the pectin formulation used is given by 30 mg of pectin dissolved in 400 pL of deionised water, thus forming the basis of the tablet placed on a sterile support. Subsequently, the sample of the substance to be tested from among those indicated was inoculated, dissolved in a growth medium in a preferred mixture of linseed and hemp oil or in linseed oil alone. The pectin tablets were maintained at a temperature of -20°C to enable them to solidify. The final solution is given by a pectin-sample w / w ratio of 20%.

[0134] The stimulations were carried out at 2h, 4h, 6h, 24h and 48h (only for iron), comparing the stimulation in individual form with the respective pectin samples. Specifically, the samples analysed were:

[0135] Sample dissolved in medium;

[0136] Sample dissolved in Q;

[0137] Sample dissolved in linseed;

[0138] Whole pectin tablet with sample dissolved in medium;

[0139] Whole pectin tablet with sample dissolved in Q;

[0140] Whole pectin tablet with sample dissolved in linseed; METHOD A.1. It entails the same preparation as Method A but before the stimulation the tablet was crushed by means of a pharmaceutical bench potter. The powder obtained was used for the stimulations. Specifically, the samples analysed were:

[0141] Sample dissolved in medium;

[0142] Sample dissolved in Q;

[0143] Sample dissolved in linseed;

[0144] Whole-fractionated pectin tablet with sample dissolved in medium;

[0145] Whole-fractionated pectin tablet with sample dissolved in Q;

[0146] Whole-fractionated pectin tablet with sample dissolved in linseed;

[0147] METHOD B. A method was devised to prepare the sample in “fractionated” pectin, thus reducing the excipient function of the pectin. Precisely, 1 mg of pectin was weighed and dissolved in 200 pL of pure deionised water to form a 10% pectin solution. Subsequently, the 200 pL of aqueous pectin solution were mixed directly with 10 mg of sample, thereby forming a solution with a 2% ratio (w / w) between the pectin solution and the sample, which was also combined with pure linseed oil, thus forming a final 2% solution of the sample and pectin (w / w), with 1 .5% linseed oil or a preferred mixture of linseed and hemp oil (Q).

[0148] Specifically, the samples analysed were:

[0149] Sample dissolved in medium;

[0150] Sample dissolved in Q;

[0151] Sample dissolved in linseed;

[0152] Fractionated pectin tablet with sample dissolved in medium;

[0153] Fractionated pectin tablet with sample dissolved in Q;

[0154] Fractionated pectin tablet with sample dissolved in linseed;

[0155] Sample preparation 2

[0156] The samples were prepared as shown above. Pectins derived from citrus fruit (Dynol) and apple (Dyno 2) were used.

[0157] The two different pectins were dissolved “hot”, 2-5 grams of pectin in 100 ml of water brought to a temperature of 35-40°C under mechanical stirring, or “cold”, i.e. 2-5 grams of pectin in 100 ml of water brought to a temperature of 15-25°C under mechanical stirring. A comparison was made between different preparation methods associated with the Dyno technology with two different pectins, a citrusbased one (Dyno 1) and a second apple-based one (Dyno 2), dissolved cold and hot on the following substances: D-chiro-lnositol (DCI) and potassium chloride (KCI).

[0158] Sample preparation 3

[0159] Following the identification of the Dyno Technology preparation method, some preliminary analyses are reported below on the beneficial impact of the Dyno Technology not only at the intestinal level but also on the target of interest, as it amplifies the effectiveness of the substance / mineral of interest:

[0160] Mulberry Extract combined with tryptophan (mulberry PLUS Dyno and DynQ) and Triple Iron Dyno combined with tryptophan.

[0161] Statistical analysis

[0162] The results are expressed as the mean ± SD of at least 5 biological replicas for every experimental protocol and every replica was repeated 3 times for every experimental protocol. Statistical comparisons between groups were performed using one-way ANOVA with a Bonferroni post hoc test or Mann-Whitney U test, depending on the cases, using GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA). A value of p<0.05 was considered statistically significant. All the other data of each experimental protocol were normalised to the control values in percentage terms (defined as 0%).

[0163] Results

[0164] Evaluation of the biological effect of pectin alone

[0165] Initially, in order to better characterise the biological effect of pectin alone at the intestinal level, the cell viability and triggering of oxidative stress after stimulation with pectin alone were described, both with regard to the formulation described in Method A and the one associated with Method B. As may be seen in figure 1 , none of the forms of pectin showed statistically significant biological effects either in terms of modulating cell viability or in terms of oxidative stress.

[0166] Iron

[0167] Iron was tested at the intestinal level using the two methods of preparing the pectin formulation. Specifically, the aim of the study was to prepare, via Method A, a layered tablet consisting of three layers (“Triple Iron”), each of which was inoculated with an iron-based solution: outer layer, iron fumarate (18pM); intermediate layer, iron pidolate (17pM); inner layer, iron bisglycinate (15pM). The Triple Iron was prepared by also adding in combination vitamin C, which allows the iron to be fixed and its absorption to be modulated. Furthermore, the tablet was tested both in the whole form (Method A) and in the whole-fractionated form (Method A.1 ), as well as in the fractionated form (Method B) before the stimulation. As may be observed in figure 2, comparing the pectin samples with the iron bisglycinate in single form as reference, Triple Iron demonstrated to maintain and increase cell viability and to inhibit oxidative stress compared to the corresponding sample dissolved in the medium (p<0.05). The sample in combination with pectin and vitamin C, in all methods, gave the best cell viability values compared to the corresponding samples not associated with pectin (p<0.05), showing a similar trend over time with a time-dependent increase in ROS production. The best results in terms of cell viability and lower oxidative stress were recorded for the sample in the fractionated formulation of Method B.

[0168] Analysing the absorption kinetics at the intestinal level in terms of extra- and intracellular iron, as may be seen in figure 3, the sample in combination with pectin demonstrated a higher absorption rate at the intestinal level compared to the corresponding samples not in pectin (p<0.05). The absorption kinetics was improved and modified compared to the reference iron bisglycinate on its own. A comparison shows that the best results are associated with the sample in pectin in combination with vitamin C, specifically in the Triple Iron prepared with Method B, compared to the corresponding samples prepared with Method A and A.1 , as it maintained good levels also after 6h-24h-48h of stimulation. At the intestinal level, a part of the formulation is retained and incorporated into intestinal cells. The non-absorbed portion, also the larger one, remains outside the cells for the whole period of stimulation (from 1 to 6h); at 24 and 48h the extracellular iron portion is reduced, suggesting that within those timeframes the extracellular iron portion is captured and introduced into the cells.

[0169] The TEER analysis confirms the better role of the samples in combination with pectin and also in the presence of vitamin C, supporting the absorption thereof. Furthermore, the analyses of ZO-1 (which mediates adhesion), Claudin (which maintains the structure) and Occludin (which contributes to stabilisation) confirm proper intestinal functionality. In the Triple Iron with vitamin C in fractionated pectin (Method B), one observes the best results in terms of maintaining the integrity and functionality of the intestinal epithelium in vitro (figure 4). Palmitoylethanolamide (PEA) Palmitoylethanolamide (PEA) was tested at the intestinal level using the two methods to prepare the PEA formulation with pectin and linseed oil and the preferred mixture of linseed and hemp oil ( ). By way of comparison, in figure 5, PEA dissolved in Q showed to maintain and increase cell viability and inhibit oxidative stress compared to the corresponding sample dissolved in the medium (p<0.05). The sample dissolved in Q in combination with pectin, in Method A and Method B, gave the best cell viability values compared to the corresponding samples not associated with pectin (p<0.05), showing a similar trend over time with a time-dependent increase in ROS production. The best results in terms of cell viability and lower oxidative stress were recorded in the tablet sample of pectin maintained whole, even though not very different compared to the fractionated form.

[0170] By way of comparison, as may be seen in figure 6, the sample dissolved in Q in combination with pectin demonstrated a higher absorption rate at the intestinal level compared to the corresponding samples not in pectin (p<0.05). The absorption kinetics is improved and modified compared to PEA, PEA linseed and PEAQ not in pectin, as optimal, constant absorption is maintained also at 6h and 24h. Comparatively, the best results are associated with the tablet sample in whole pectin.

[0171] The TEER analysis confirms the better role of the samples dissolved in linseed oil and dissolved in Q, supporting the absorption thereof. Furthermore, the analyses of ZO-1 (which mediates adhesion), Claudin (which maintains the structure) and Occludin (which contributes to stabilisation) confirm proper intestinal functionality. In the sample dissolved in Q in whole pectin, one observes the best results in terms of maintaining the integrity and functionality of the intestinal epithelium in vitro (figure 7).

[0172] Gastrodia

[0173] Gastrodia was tested at the intestinal level using the two methods (A and B) to prepare the Gastrodia formulation with pectin and linseed oil or dissolved in Q. By way of comparison, in figure 8, Gastrodia dissolved in Q showed to maintain and increase cell viability and inhibit oxidative stress compared to the corresponding sample dissolved in the medium and the corresponding sample dissolved in linseed (p<0.05). The sample dissolved in Q in combination with pectin, with both methods (A and B), gave the best cell viability values when compared to the corresponding samples not associated with pectin (p<0.05), showing a similar trend over time with a time-dependent increase in ROS production. By way of comparison, as may be seen in figure 9, the sample dissolved in Q in combination with pectin demonstrated a higher absorption rate at the intestinal level compared to the corresponding samples not in pectin or in linseed oil (p<0.05). The absorption kinetics was not modified with Method A but improved compared to Gastrodia and Gastrodia Q not in pectin, as optimal, constant absorption was maintained also at 6h and 24h. The absorption kinetics, by contrast, showed to be modified with Method B compared to Gastrodia and GastrodiaQ not in pectin, and showed a plateau phase between 4h and 6h with constant absorption also at 24h.

[0174] The TEER analysis confirms the better role of the samples dissolved in linseed oil or dissolved in Q, supporting the absorption thereof. Furthermore, the analysis of ZO-1 (which mediates adhesion), Claudin (which maintains the structure) and Occludin (which contributes to stabilisation) confirm proper intestinal functionality. In the sample dissolved in Q in fractionated pectin, one observes the best results in terms of maintaining the integrity and functionality of the intestinal epithelium in vitro compared to the sample prepared with method A (figure 10).

[0175] D-Mannose

[0176] The D-Mannose sample was tested at the intestinal level using the two methods (A and B) to prepare the D-Mannose formulation with pectin and linseed oil or dissolved in Q. By way of comparison, in figure 11 , D-Mannose dissolved in Q showed to maintain and increase cell viability and inhibit oxidative stress compared to the corresponding sample dissolved in the medium or in linseed oil (p<0.05). The sample dissolved in Q in combination with pectin, in both methods (A and B), gave the best cell viability values compared to the corresponding samples not associated with pectin (p<0.05), showing a similar trend over time with a time-dependent increase in ROS production. The best results in terms of cell viability and lower oxidative stress were recorded for the sample dissolved in Q in pectin prepared following method B.

[0177] By way of comparison, as may be seen in figure 12, the sample dissolved in Q in combination with the pectin demonstrated a higher absorption rate at the intestinal level compared to the corresponding samples not in pectin or in linseed oil (p<0.05). The absorption kinetics showed to be improved and modified compared to D-Mannose and D-MannoseQ not in pectin, as optimal, constant absorption is maintained also at 6h and 24h, especially in the sample dissolved in Q in pectin and the sample dissolved in linseed in pectin following method B.

[0178] The TEER analysis confirms the better role of the samples dissolved in linseed oil and dissolved in Q, supporting the absorption thereof. Furthermore, the analyses of ZO-1 (which mediates adhesion), Claudin (which maintains the structure) and Occludin (which contributes to stabilisation) confirm proper intestinal functionality. In the sample dissolved in Q in fractionated pectin prepared with Method B, one observes the best results in terms of maintaining the integrity and functionality of the intestinal epithelium in vitro (figure 13).

[0179] Ginger

[0180] Ginger was tested at the intestinal level using the two methods (A and B) to prepare the ginger formulation with pectin and linseed oil or in Q oil. By way of comparison, in figure 14, ginger dissolved in Q showed to maintain and increase cell viability and inhibit oxidative stress compared to the corresponding sample dissolved in the medium (p<0.05). The sample dissolved in Q in combination with pectin, with both methods (A and B), gave the best cell viability values compared to the corresponding samples not associated with pectin or linseed oil (p<0.05), showing a similar trend over time with a time-dependent increase in ROS production. The best results in terms of cell viability and lower oxidative stress were recorded for the sample dissolved in Q with both methods (A and B).

[0181] By way of comparison, as may be seen in figure 15, the sample dissolved in Q in combination with pectin demonstrated a higher absorption rate at the intestinal level compared to the corresponding samples not in pectin or linseed oil (p<0.05). The absorption kinetics was improved and modified compared to ginger and ginger Q not in pectin, as optimal, constant absorption is maintained also at 6h and 24h. Comparatively, the best results are associated with the sample dissolved in Q in a fractionated pectin tablet, based on absorption, but a whole pectin tablet based on kinetics.

[0182] The TEER analysis confirms the better role of the samples dissolved in linseed oil or in Q oil, supporting the absorption thereof. Furthermore, the analyses of ZO-1 (which mediates adhesion), Claudin (which maintains the structure) and Occludin (which contributes to stabilisation) confirm proper intestinal functionality. In the sample dissolved in Q in fractionated pectin, the best results are observed in terms of maintaining the integrity and functionality of the intestinal epithelium in vitro (figure 16).

[0183] D-chiro-lnositol (PCI) and potassium chloride (KCI)

[0184] The TEER analysis made it possible to demonstrate an active role of DCI, dissolved both in growth medium and SimbioQ or combined with Dyno technology, in maintaining barrier integrity and functionality. The data demonstrate that the combination with SimbioQ increased the beneficial effects at the intestinal level, which were more greatly improved thanks to Dyno technology using both methods and with better effects from the hot method for both selected pectins. Better data were found in terms of pectin type for Dyno 1 (citrus, p<0.05), with good effects also for Dyno 2, improved with the hot method. Furthermore, the analyses of Claudin-1 (which maintains the structure), Occludin (which contributes to stabilisation) and ZO-1 (which mediates adhesion) confirm proper intestinal functionality in both samples (Figure 17).

[0185] Further information on intestinal absorption were obtained by analysing the passage through the intestinal barrier in order to characterise the bioavailability and permeability of the samples under examination.

[0186] DCI in combination with the SimbioQ, Dyno technologies demonstrated significant differences in terms of permeability at the intestinal level compared to DCI dissolved in growth medium already starting from 2h of stimulation (p<0.05). In all the samples, the peak of maximum absorption was detected at around 4h of stimulation.

[0187] In the case of both pectins under examination, the Dyno technology promoted a distinct increase in permeability and in the absorption rate of DCI along the intestinal barrier, with more significant data for Dyno 1 DCI compared to the other samples (p<0.05), while the statistical difference was reduced after 5h of stimulation.

[0188] In this case as well, apple pectin (Dyno 2) was enhanced through the application of the hot method (Figure 18).

[0189] The TEER analysis made it possible to demonstrate an active role of KCI, dissolved both in growth medium and SimbioQ or combined with Dyno technology, in maintaining barrier integrity and functionality. The data demonstrate that the combination with SimbioQ increased the beneficial effects at the intestinal level, which showed to be more greatly improved thanks to the Dyno technology using both methods, with better effects from the hot method for both pectins selected. Better data were found in terms of type of pectin for Dyno 2 (apple, p<0.05), with good effects also for Dyno 1 (citrus), improved by the hot method. Furthermore, the analyses of Claudin-1 (which maintains the structure), Occludin (which contributes to stabilisation) and ZO-1 (which mediates adhesion) confirm proper intestinal functionality in both samples (Figure 19).

[0190] Further information on intestinal absorption was obtained by analysing the passage through the intestinal barrier in order to characterise the bioavailability and permeability of the samples under examination.

[0191] KCI, in combination with the SimbioQ and Dyno technologies, demonstrated significant differences, in terms of permeability at the intestinal level, compared to KCI dissolved in growth medium already starting from 2h of stimulation (p<0.05).

[0192] In the case of both pectins under examination, the hot Dyno technology promoted a distinct increase in permeability and in the absorption rate of KCI along the intestinal barrier, with more significant data for Dyno 2 KCI compared to the other samples (p<0.05) only at 4h. Both forms of pectin were enhanced thanks to the hot method (Figure 20).

[0193] Mulberry extract combined with tryptophan (Mulberry PLUS Dyno and DynQ) and triple iron with tryptophan

[0194] An absorption analysis was performed to evaluate the absorption of the formulation based on Mulberry PLUS. Based on the data shown in the figure, it is possible to note that: the sample in combination with DynoQ 1 and DynoQ 2 demonstrated a higher absorption rate at the intestinal level compared to the corresponding samples prepared in Dyno 1 and Dyno 2 (p<0.05). The absorption kinetics was improved and modified compared to the preparation with DynoQ 2 (p<0.05) (Figure 21).

[0195] Analyses were performed on the production of insulin and its receptor substrates (IRS1 ) to evaluate the effects of Mulberry PLUS (mulberry with tryptophan) prepared with two different formulations of Dyno and DynoQ on insulin metabolism. The results of the IRS1 analysis indicate that all the agents under examination were capable of stimulating insulin production (p<0.05). Specifically, the DynoQ 1 and DynoQ 2 preparations were capable of enhancing this effect by activating IRS1 and increasing the production of insulin more effectively than Mulberry PLUS alone and the Dyno 1 and Dyno 2 preparations. Among all the preparations, the best as regards both parameters is DynoQ 1 (p<0.05) (Figure 22).

[0196] In order to confirm the previous data related to insulin activity, the reduction in circulating glucose was evaluated. The results of the analysis on circulating glucose indicate that all the agents under examination were capable of reducing the levels of circulating glucose (p<0.05). Specifically, the DynoQ 1 and DynoQ 2 preparations were capable of better enhancing this effect of Mulberry PLUS than the Dyno 1 and Dyno 1 preparations. DynoQ 1 showed to be the best preparation, since it reduced the circulating glucose levels, bringing them back to the level of physiological values (p<0.05) (Figure 23).

[0197] Analyses of DMT-1 and ferritin activity were caried out to assess the effects of Triple Iron prepared with two different Dyno formulations on iron metabolism. The results of the analyses indicate that all the agents under examination were capable of influencing this process (p<0.05). The effect appeared to be improved by the interaction with Tryptophan. Furthermore, the DynoQ 1 and DynoQ 2 preparations were capable of enhancing it, increasing not only the absorption of iron but also its interaction with ferritin at the enterocyte level, to a better degree than iron bisglycinate alone (p<0.05) (figure 24).

[0198] Table 1 : Analysis of absorption in the Transwell model with Caco-2 cells

[0199] Dyno technology has made it possible to improve iron absorption by further amplifying the combined effect with tryptophan without inducing any alteration compared to iron bisglycinate (p<0.05).

Claims

CLAIMS1 . A composition comprising a polysaccharide, at least one vegetable oil and at least one molecule or compound, wherein the polysaccharide is pectin, wherein the at least one vegetable oil is selected from linseed oil and hemp oil and wherein the at least one molecule or compound is an active ingredient or a nutraceutical substance.

2. The composition according to claim 1 , comprising tryptophan.

3. The composition according to claim 1 or 2, wherein the at least one vegetable oil is linseed oil.

4. The composition according to any one of claims 1 -3, comprising a mixture of linseed oil and hemp oil.

5. The composition according to claim 4, wherein the volume / volume ratio between linseed oil and hemp oil ranges between 60:40 and 95:5.

6. The composition according to claim 4 or 5, wherein the volume / volume ratio between linseed oil and hemp oil ranges between 65:35 and 85:15.

7. The composition according to any one of claims 4-6, wherein the ratio between linseed oil and hemp oil is comprised between 70:30 and 85:15.

8. The composition according to any one of claims 4-7, wherein the ratio between linseed oil and hemp oil is comprised between 70:30 and 80:20.

9. The composition according to any one of claims 4-8, wherein the ratio between linseed oil and hemp oil is 75:25.

10. The composition according to any one of claims 1-9, wherein tryptophan is present in a concentration of between 0.05 and 2% weight / weight (w / w) relative to the weight of the composition, preferably between 0.8 and 1% w / w, more preferably between 0.1 and 0.5% w / w.

11. The composition according to any one of claims 1-9, wherein the polysaccharide is present in a concentration of between 0.5 and 30% weight / weight (w / w) relative to the weight of the composition, preferably between 5 and 25% w / w, more preferably between 10 and 20% w / w.

12. The composition according to any one of claims 1-11 , wherein the linseed oil is present in a concentration of between 0.5 and 15% weight / weight (w / w) relative to the weight of the composition, preferably between 1 and 10% w / w, more preferably between 1 and 5% w / w.

13. The composition according to any one of claims 1 -12, wherein the mixture of linseed oil and hemp seed oil is present in a concentration of between 0.5 and 15 % weight / weight (w / w) relative to the weight of the composition, preferably between 1 and 10% w / w, more preferably between 1 and 5% w / w.

14. The composition according to any one of claims 1-13, wherein said at least one molecule or compound is selected from: ions, preferably iron, minerals, preferably magnesium, N- acylethanolamines, potassium chloride, preferably palmitoylethanolamide (PEA), sugars, preferably D-mannose, vitamins preferably Vitamin D and Vitamin C, resins, preferably propolis, amino acids, preferably N-acetyl-cysteine, poly-olicarbocyclics, preferably D-chiro-lnositol, fungi and mycetes, preferably Reishi, phenols and polyphenols, preferably Gastrodia elata Blume and lycopene and curcumin, flavonoids, preferably resveratrol, isoflavonoids, bioflavonoids, phytoestrogens, preferably daidzein, carotenoids, preferably astaxanthin, medicinal herbs, plant extracts, preferably ginger and extracts thereof, gastrodia and extracts thereof, lactic acid bacteria, lactoferrin, melatonin, coenzyme Q10, mulberry extract and combinations thereof.

15. The composition according to any one of claims 1-14, formulated for oral use, preferably as a lozenge, tablet, granules, pill, capsule, hard-shelled capsule, or syrup.

16. The composition according to any one of claims 1-15, for use as a medicament.

17. The composition according to any one of claims 1-15, for use in the prevention or treatment of a pathology selected from: a neurological, immune system, ophthalmic, urogenital, urological and gynaecological, cardiovascular, respiratory, gastrointestinal, osteoarticular, cutaneous, and muscular pathology, malabsorption syndrome and / or metabolic pathologies.

18. Use of the composition according to any one of claims 1-15 as a nutraceutical.

19. Use of the composition according to claim 18 as a dietary supplement, to increase the absorption or metabolism or bioavailability of compounds or molecules preferably with low bioavailability, more preferably of orally administered compounds.

20. A dietary supplement comprising the composition according to any one of claims 1 -15.

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