NANOPARTICLES OF COCOA EXTRACT AND USE THEREOF AS ANTIOXIDANTS

Technical Field

The present invention relates to chitosan-based nanoparticles encapsulating an extract of cocoa hulls and/or nibs comprising polyphenols, the process for preparing them and the use thereof as antioxidant agents in the prevention of cellular oxidative stress and/or for the preparation of nutraceutical, pharmaceutical, cosmetic or food formulations.

Prior Art

Polyphenols are a particularly interesting family of molecules, as they are characterised by carrying out a large variety of biological activities that are potentially beneficial for human health, first and foremost antioxidant activity. Polyphenols are in fact potent natural antioxidants and are naturally present in fruit and vegetables and, in larger quantity, in tea and wine, red wine in particular. Another important source of polyphenols is represented by cocoa ( Theobroma cacao L), in particular cocoa beans, whose content of polyphenols can vary depending on the type of beans or depending on the processing of the finished product. For example, as reported in the paper by Adamson, G. E. et. al. (“ HPLC method for the quantification of procyanidins in cocoa and chocolate samples and correlation to total antioxidant capacity’, Journal of Agricultural and Food Chemistry, 47, 4184-4188), polyphenols can be found in an amount comprised between 3.3 mg/g and 65 mg/g in cocoa powder whilst the amount thereof can range between 1.7 mg/g and 36.5 mg/g in dark chocolate. The majority of polyphenolic (or phenolic) compounds present in cocoa are represented by flavonols, which include catechins, epicatechins, procyanidins and proanthocyanidins or tannins whose molecular weight can vary greatly, ranging from a simple monomer to a long-chain polymer. These substances not only possess a high antioxidant capacity but also multiple biological and pharmacological activities that induce an antimicrobial, anti-inflammatory and anticariogenic effect, as described, for example, in patent EP 1304047 regarding extracts of cocoa bean hulls with inhibitory effects on carcinogenesis. As already mentioned, such substances thus play a fundamental role in eliminating free radicals, which are responsible for peroxidation on a cellular level, thus carrying out activities that are potentially beneficial for the health of the human body.

As far as these activities are concerned, the most studied at present is undoubtedly that on the cardiovascular system. Various epidemiological studies suggest, in fact, that a regular consumption of foods and beverages rich in polyphenols, in particular cocoa, can be associated with a reduction in the risk of various pathological conditions ranging from hypertension to coronary heart disease to heart attack or dementia. However, cocoa, and particularly the foods containing it, undergo a large number of processes before being eventually consumed; these processes have the purpose of refining, storing, cooking or preserving the food and can lead to a substantial loss of polyphenols or conversion into phenolic forms possessing less antioxidant activity. Since polyphenolic substances are contained in greater amounts in cocoa hulls and/or nibs, in recent years the study of the content and pharmacological activity of the polyphenols present in cocoa has evolved alongside the development of techniques for extracting and recovering such polyphenols for the production of extracts and the use thereof in various pharmacological therapies. Obtaining extracts from cocoa hulls and/or nibs provides advantages not only in terms of polyphenol content but also in terms of costs and environmental impact, since hulls and/or nibs represent a by-product of the agro-industrial processing of cocoa, which is generated in large amounts during the production of chocolate and is however generally discarded, with consequent disposal costs and environmental contamination. Generally, as described, for example, in patents US4156030 or EP1728434, in the processes for producing extracts from cocoa hulls known in the prior art, after harvesting, fermentation and drying, the cocoa beans are reduced into medium-sized pieces, giving rise to so-called cocoa nibs. A system of sieves and suction devices then separates the nibs from the outer hulls of the cocoa beans. The hulls are then treated with solvents, such as, for example, acidified ethanol or acetone, in order to obtain water-soluble extracts rich in the substances listed above. However, the largest problem present in the prior art associated with the use of polyphenols contained in cocoa or in the extracts thereof as antioxidant agents in therapy is their low bioavailability. In fact, the low absorption by the intestine, associated with their oxidation in the gastrointestinal tract and high metabolism in the liver, make it highly improbable that high concentrations of these substances can be found for any length of time in the body after they have been ingested and that they may then reach the bloodstream, i.e. the site of action, in amounts which are effective for the performance of their activity.

This problem is solved by the present invention, which provides water-soluble extracts of cocoa hulls and/or nibs encapsulated within chitosan-based nanoparticles. The nanoparticles of the present invention make it possible, in fact, to extend the gastrointestinal residence time of the polyphenols contained in said extracts while simultaneously decreasing the influence of the natural mechanisms of intestinal clearance. Furthermore, given their small size, the use of the nanoparticles developed by the Applicant makes it possible to increase the surface available for interaction with the biological target and to penetrate into tissues through capillaries, so that they are directly internalised by cells and their absorption is thus promoted. Object of the invention

The present invention relates to chitosan-based nanoparticles encapsulating a water-soluble extract of cocoa hulls and/or nibs, comprising polyphenols and a process for the preparation thereof.

Said polyphenols are preferably selected in the group consisting of: /V-caffeoyl aspartate, catechin, epicatechin, quercetin 3-O-glucoside, quercetin 3-O-arabinoside, polyphenols belonging to the procyanidin class, preferably B- and C-type procyanidins, more preferably procyanidin Bi and procyanidin B ₂ , and a combination thereof. The nanoparticles of the present invention appear as nanoparticles in an aqueous suspension or nanoparticles in a powder to be reconstituted in an aqueous solvent and they have a diameter whose average dimensions is comprised in the order of hundreds of nanometres.

The present invention further relates to the use of said nanoparticles for the preparation of nutraceutical, pharmaceutical, cosmetic or food formulations. The nanoparticles can also be used in the prevention of cellular oxidative stress and in the treatment or in the prevention of the risk of pathological conditions of the cardiovascular system of an individual. The invention also relates to a nutraceutical, pharmaceutical, cosmetic or food composition comprising chitosan-based nanoparticles encapsulating a water-soluble extract of cocoa hulls and/or nibs.

Brief description of the drawings

Figure 1 shows the HPLC-ESI-MS/MS profile of extracts of nibs and hulls of cocoa of the Costa Rica variety (respectively graphs a) and b)) and of hulls of cocoa of the Madagascar variety (graph c)), obtained as described in Example 1 , paragraph 1 .3.

Figure 2 shows the dimensional analysis performed by means of the Dynamic Light Scattering (DLS) technique on nanoparticles in an aqueous suspension encapsulating an extract of hulls of cocoa of the Venezuela Barinas variety obtained as per Example 1 , paragraph 1 .5.

Figure 3 shows the dimensional analysis performed by means of the Dynamic Light Scattering (DLS) technique on nanoparticles in an aqueous suspension encapsulating an extract of hulls of cocoa of the Madagascar variety obtained as per Example 1 , paragraph 1.5.

Figure 4 shows the dimensional analysis performed by means of the Dynamic Light Scattering (DLS) technique on nanoparticles in an aqueous suspension encapsulating an extract of nibs of cocoa of the Costa Rica variety obtained as per Example 1 , paragraph 1.5. Figure 5 shows the dimensional analysis performed by means of the Dynamic Light Scattering (DLS) technique on nanoparticles in an aqueous suspension encapsulating an extract of hulls of cocoa of the Costa Rica variety obtained as per Example 1 , paragraph 1.5. Figure 6 shows the dimensional analysis performed by means of the Dynamic Light Scattering (DLS) technique on nanoparticles in an aqueous suspension encapsulating an extract of hulls of cocoa of the Costa Rica variety obtained as per Example 1 , paragraph 1.7. Figure 7 shows the dimensional analysis performed by means of the Dynamic Light Scattering (DLS) technique on nanoparticles in powder form redispersed in a simulated gastric fluid (SGF) encapsulating an extract of hulls of cocoa of the Costa Rica variety.

Figure 8 shows two STEM images (magnification 40000X) of nanoparticles encapsulating an extract of hulls of cocoa of the Costa Rica variety (image a)) and nanoparticles encapsulating a solution of procyanidin B ₃ (“procyanidin B ₃ standard”) (image b)).

Figure 9 shows the trend over time of the permeation of extract through isolated rat intestinal tissue. The trend is shown in the case of a free extract (i), in the case of said extract encapsulated in the nanoparticles of the invention (ii), in the case of a solution of procyanidin B ₃ in a free form (iii) and in the case of said solution encapsulated in the nanoparticles of the invention (iv).

Figure 10 shows the cell viability test after 2 hours of treatment of umbilical vein endothelial cells (FIUVECs) with a free extract (COCOA) and with the nanoparticles of the invention encapsulating said extract (COCOA NPs).

Figure 1 1 shows the cell viability test after 24 hours of treatment of umbilical vein endothelial cells (FIUVECs) with a free extract (COCOA) and with the nanoparticles of the invention encapsulating said extract (COCOA NPs); ^* P<0.05, ^** P<0.01 , ^*** P<0.001 .

Figures 12 A and 12 B show the basal production of ROS of umbilical vein endothelial cells (FIUVECs) in the case of untreated FIUVECs (CN) and in the case of FIUVECs incubated for 2 hours (12 A) and 24 hours (12 B) with a free extract (COCOA) and with the nanoparticles of the invention encapsulating said extract (COCOA NPs); ^**** P<0.0001.

Figures 13 A and 13 B show the basal production of ROS of umbilical vein endothelial cells (FIUVECs) in the case of untreated FIUVECs (CN) and in the case of FIUVECs treated with H2O2 and pre-incubated for 2 hours (13 A) and 24 hours (13 B) with a free extract (COCOA) and with the nanoparticles of the invention encapsulating said extract (COCOA NPs); ^* P<0.05, ^** P<0.01 , ^*** P<0.001 , ^**** P<0.0001.

Detailed description of preferred embodiments of the invention

The term “polyphenols” or “polyphenolic substances” or “polyphenolic compounds” or “phenolic compounds” means a class of organic molecules comprising flavonols, catechins, epicatechins, procyanidins and proanthocyanidins. These molecules are characterised by the presence of a number of phenolic groups which are associated in more or less complex structures ranging from substances comprising a single monomer or only a few monomers to molecules with a high molecular weight.

The term “cocoa bean” means the seeds of the cocoa plant called Theobroma cacao covered by the hull.

The term“cocoa nibs” means the product obtained by reducing cocoa beans into medium sized pieces after harvesting, fermentation and drying.

The term“raw cocoa" or“raw cocoa beans” means cocoa or cocoa beans that have not undergone the roasting process.

The term“cocoa extract” refers indistinctly to an extract deriving from cocoa hulls and/or nibs.

The term“tree extract” or“extract in a free form” means a water-soluble extract of cocoa hulls and/or nibs that is not encapsulated within nanoparticles or any other carrier. This terminology refers, therefore, to an aqueous solution or suspension of the non-encapsu!ated water-soluble extract.

The term“variety”,“variety of cocoa beans” or“cocoa variety”, means the type of cocoa beans identified based on their geographical origin.

The present invention relates to chitosan-based nanoparticles encapsulating a water-soluble extract of cocoa hulls and/or nibs, wherein the extract comprises polyphenols. Preferably, said water-soluble extract of cocoa hulls and/or nibs encapsulated in the nanoparticles according to the present invention comprises polyphenols selected in the group consisting of: /V-caffeoyl aspartate, catechin, epicatechin, quercetin 3-O-glucoside, quercetin 3-0- arabinoside, polyphenols belonging to the procyanidin class and a combination thereof. More preferably, said polyphenols are polyphenols belonging to the procyanidin class, preferably B- and C-type procyanidins, more preferably procyanidin Bi and procyanidin B ₂ . According to one embodiment, said procyanidins are selected from procyanidin dimers and procyanidin trimers; even more preferably, said procyanidins are selected in the group consisting of: procyanidin trimer I, procyanidin trimer II, procyanidin trimer III, procyanidin trimer V, procyanidin trimer VI, procyanidin trimer VII, procyanidin dimer I, procyanidin dimer II, procyanidin dimer III and a combination thereof. In one embodiment of the invention, said water-soluble extract comprises a total amount of polyphenols comprised between 20 and 80 mg/g, preferably between 40 and 70 mg/g, more preferably between 50 and 70 mg/g, even more preferably between 60 and 70 mg/g relative to the total weight of the extract. The chitosan-based nanoparticles encapsulating a water-soluble extract of cocoa hulls and/or nibs according to the present invention preferably do not comprise stabilisers, cross-linking agents or other excipients commonly used in the sector. Said stabilisers, cross-linking agents or other excipients are preferably selected from polymers comprising acidic groups and derivatives thereof. More preferably, said stabilisers, cross-linking agents or other excipients are selected in the group consisting of: hyaluronic acid, tripolyphosphate, pectin, xanthan gum, gum arabic, gellan gum, alginic acid, fucoidan, compounds belonging to the carrageenan class and a combination thereof. Said total amount and the type of polyphenols depends on the variety of cocoa beans used and the starting material from which the extract encapsulated in the nanoparticles of the invention is obtained. Said starting material comprises cocoa hulls and/or nibs deriving from raw cocoa beans. Therefore, the water- soluble extract of cocoa hulls and/or nibs is more precisely a water-soluble extract of raw cocoa hulls and/or nibs. For the purposes of the present invention, these two expressions are used and interchangeable synonyms. In a preferred embodiment of the invention, the starting material used is cocoa hulls. The total amount and type of polyphenols, furthermore, is also closely connected to the origin of the cocoa beans. In fact, not only does each region have a different microclimate and soils, but the techniques for cultivating, harvesting and processing cocoa beans can also vary from country to country, thus making all varieties of cocoa beans, even if they belong to the same subspecies (Forastero, Criollo or Trinitario cocoa), profoundly different and distinguishable from one another. The variety of cocoa beans used is selected in the group defined by the geographical origin of the cocoa beans themselves and consisting of: Costa Rica, Jamaica, Madagascar, Ecuador bio, Venezuela porcelana, Ghana, Trinidad, Tobago, Grenada and Venezuela Barinas varieties. Among these, the cocoa beans coming from Ghana belong to the Forastero cocoa ( Theobroma cacao sphaerocarpum) subspecies, whereas the others, coming from Costa Rica, Jamaica, Madagascar, Ecuador bio, Venezuela porcelana, Trinidad, Tobago, Grenada, and Venezuela Barinas, belong to the Trinitario cocoa subspecies, a hybridisation of the Criollo cocoa and Forastero cocoa subspecies. The variety of cocoa beans used is preferably selected from the cocoa varieties whose extract deriving from hulls and/or nibs has the highest content of total polyphenols. According to a particularly preferred embodiment of the invention, the variety of cocoa beans used is preferably selected from the cocoa varieties whose extract deriving from hulls and/or da nibs has polyphenols selected in the group consisting of: /V-caffeoyl aspartate, catechin, epicatechin, quercetin 3-O-glucoside, quercetin 3-O-arabinoside, polyphenols belonging to the procyanidin class and a combination thereof. More preferably said polyphenols are polyphenols belonging to the procyanidin class, preferably B- and C- type procyanidins, more preferably procyanidin Bi and procyanidin B ₂ . According to one embodiment, said procyanidins are selected from procyanidin dimers and procyanidin trimers; even more preferably said procyanidins are selected in the group consisting of: procyanidin trimer I, procyanidin trimer II, procyanidin trimer III, procyanidin trimer V, procyanidin trimer VI, procyanidin trimer VII, procyanidin dimer I, procyanidin dimer II, procyanidin dimer III and a combination thereof. Said varieties are preferably selected in the group consisting of the Costa Rica, Madagascar and Venezuela Barinas varieties. In a preferred embodiment, the cocoa variety used is the Costa Rica variety, whose extract of hulls and/or nibs comprises a high amount of total polyphenols, preferably comprised between 40 mg/g and 70 mg/g, more preferably between 50 and 70 mg/g, even more preferably between 60 and 70 mg/g, relative to the total weight of the extract, said polyphenols being preferably selected in the group consisting of: /V-caffeoyl aspartate, catechin, epicatechin, quercetin 3-O-glucoside, quercetin 3-O-arabinoside, procyanidin trimer I, procyanidin trimer II, procyanidin trimer III, procyanidin trimer V, procyanidin trimer VI, procyanidin trimer VII, procyanidin dimer I, procyanidin dimer II, procyanidin dimer III and a combination thereof.

The present invention also relates to a process for preparing the nanoparticles encapsulating a water-soluble extract of cocoa hulls and/or nibs, comprising the steps of:

a) subjecting raw cocoa beans to grinding until obtaining a homogenous fragmented starting material comprising hulls and cocoa nibs;

b) performing an extraction in an organic solvent, preferably in acetone, and eliminating the organic solvent used in the extraction, thereby obtaining a water-soluble dry extract of cocoa hulls and/or nibs;

c) suspending commercial chitosan (food-grade) in water, acidifying by adding an organic and/or inorganic acid and maintaining under magnetic stirring for 12-16 hours;

d) preparing an aqueous solution of the extract of step b) and adding it to the suspension obtained in step c), under continuous stirring and at room temperature.

In one embodiment of the invention said process for preparing the nanoparticles comprises a step a’) which provides for the separation of the hulls from the raw cocoa nibs by means of conventional techniques (e.g. sieves and suction devices). In this case, the subsequent steps b)-d) of extracting the polyphenolic compounds and synthesising the nanoparticles are therefore carried out separately on the hulls and cocoa nibs so as to obtain, respectively, nanoparticles encapsulating an extract of cocoa hulls and nanoparticles encapsulating an extract of cocoa nibs. In one embodiment, before the extraction in an organic solvent of step b), the starting material preferably undergoes washing with an organic solvent, for example hexane, preferably repeated several times (for example 4 times). The purpose of the washing is to degrease the starting material. Step b) of extraction in an organic solvent can be repeated several times, for example 4 times, so as to maximise the content of polyphenols in the extract.

The organic and/or inorganic acid used in step c) can be for example HCI. The process for preparing the nanoparticles of the present invention is a process that exploits an ionotropic cross-linking reaction. This process provides advantages which are not only connected to the extreme simplicity and speed of execution, but are also due to the mild reaction conditions (in particular in terms of temperature and pH), as the process is based on the self-assembly, into nanoparticles, of the cocoa extract with chitosan, without the addition of stabilisers, cross-linking agents or other excipients and without the use of organic solvents. In fact, the nanoparticles of the present invention form spontaneously when a chitosan solution (step c)) is placed in contact with an aqueous solution of the extract (step d)). Said stabilisers, cross- linking agents or other excipients are preferably selected from polymers comprising acidic groups and derivatives thereof. More preferably, said stabilisers, cross-linking agents or other excipients are selected in the group consisting of: hyaluronic acid, tripolyphosphate, pectin, xanthan gum, gum arabic, gellan gum, alginic acid, fucoidan, compounds belonging to the carrageenan class and a combination thereof. In an alternative embodiment, the process for preparing the nanoparticles encapsulating an extract of cocoa hulls and/or nibs according to the present invention is a process wherein after step b), as an alternative to steps c) and d), the water-soluble dry extract of cocoa hulls and/or nibs is mixed with commercial chitosan in powder and the dry mixture thus obtained is suspended in water under continuous stirring and at room temperature (steps c’) and d’)), until obtaining the nanoparticles of the invention by self-assembly in an aqueous suspension. Said embodiment, as it envisages the self-assembly of nanoparticles in an aqueous suspension starting from a mixture of powders (dry cocoa extract and commercial chitosan powder), proves to be an extremely simple, rapid process. With this process it is also reasonable to be able to envisage the possibility of forming the nanoparticles of the invention directly in situ once the components of the dry mixture, after ingestion of said mixture by an individual, enter into contact with the physiological fluids of the individual him/herself. Without wishing to be bound to a specific theory, the Applicant has nonetheless found that it is precisely thanks to the type of polyphenols present in the extract deriving from cocoa hulls and/or nibs as previously described that it is possible to obtain self-assembly (in an aqueous solution and without the addition of stabilisers, cross-linking agents or other excipients and without the use of organic solvents) of nanoparticles having the desired dimension and encapsulation efficiency values. Another aspect that may possibly be considered relevant for the purpose of obtaining the aforesaid self-assembly is the total amount of polyphenols present in the extract, which, as shown in the Examples section, can potentially influence the formation of nanoparticles having the desired dimension and encapsulation efficiency values. The nanoparticles obtained following step d) or d’) are nanoparticles in an aqueous suspension. The nanoparticles of the present invention can be used in that form or else in a dry form, that is, in the form of a powder to be reconstituted in an aqueous solvent. The nanoparticles in a dry form according to the present invention can be obtained by means of a subsequent step e) of eliminating the solvent by means of any technique known in the art. The nanoparticles in a dry form, that is, in the form of a powder to be reconstituted in an aqueous solvent, are preferably obtained by means of the lyophilisation technique. According to one embodiment, the nanoparticles encapsulating a water-soluble extract of cocoa hulls and/or nibs according to the present invention have a diameter whose dimensions are comprised in the order of hundreds of nanometres. Said dimensions have preferably comprised between 100 and 600 nm, more preferably between 200 and 400 nm, even more preferably between 300 and 400 nm, measured by means of the Dynamic Light Scattering (DLS) technique. The nanoparticles of the invention further have a polydispersity index (PDI) that is preferably comprised between 0.1 and 0.8, preferably between 0.1 and 0.4. These dimensions have shown to be the preferred dimensions for the purposes of the present invention, since it enables the amount of extract encapsulated within the nanoparticles to be effectively balanced while simultaneously maintaining the desired characteristics and technical effects which are closely linked to the nanometric dimensions, e.g. the large surface available for interaction with the biological target, the ability to penetrate into tissues through capillaries, and the possibility, for the nanoparticles, to be internalised by cells, something that does not occur in the case of larger-sized particles (e.g. particles with dimensions in the order of pm). According to one embodiment, the nanoparticles encapsulating a water-soluble extract of cocoa hulls and/or nibs according to the present invention are characterised by having a zeta potential value (measured by means of the light-scattering technique) comprised between 10 and 25 mV, preferably between 15 and 20 mV. The nanoparticles of the present invention, furthermore, are preferably stable in an aqueous solvent and in a wide pH range, preferably comprised between 1 and 8, and in particular at acidic pH levels, preferably at acidic pH levels comprised between 1 and 5.

This represents a considerable advantage since, as also demonstrated in the Examples section in relation to the stability experiments on nanoparticles in a simulated gastric fluid, the nanoparticles encapsulating the extract containing polyphenols according to the present invention are capable of remaining stable even at acidic pH levels and of protecting, therefore, the encapsulated extract from the mechanisms of degradation in the gastrointestinal tract, thus also promoting their absorption by intestinal tissue. The nanoparticles encapsulating an extract of cocoa hulls and/or nibs according to the present invention in fact show a greater ability to pass through the intestinal barrier compared to the free extract, thus enabling a greater permeation of the extract, and hence of the polyphenols contained therein, through intestinal tissue. The nanoparticles of the present invention are thus effective both in promoting intestinal absorption of the cocoa extract encapsulated therein and in protecting said extract from degradation by gastric and intestinal fluids.

Without wishing to be bound by any mechanism of action, the experiments reported in the Examples section have shown that the extract of cocoa hulls and/or nibs comprising polyphenols encapsulated in the nanoparticles of the present invention does not decrease cell viability, nor does it cause an increase in the production of reactive oxygen species (ROS) at any of the tested total polyphenol concentrations. Said experiments further demonstrated that the nanoparticles of the present invention encapsulating an extract of cocoa hulls and/or nibs comprising polyphenols are capable of protecting cells from oxidative stress in a manner comparable to the case of a free extract. Therefore, the encapsulation of said extract within the nanoparticles according to the present invention does not affect the antioxidant effectiveness of the polyphenols contained in the extract itself, said polyphenols being preferably as previously described. The nanoparticles of the present invention therefore not only show a greater ability in promoting the permeation of the extract contained therein through intestinal tissue, but also enable said extract to be effectively protected in the gastrointestinal tract and, once it is in contact with cells, to behave similarly to a free extract, thus providing all the effects attributed to the presence of polyphenols, without the disadvantages connected to the use of a non-encapsulated extract, such as, for example, phenomena connected to degradation in the gastrointestinal tract, high liver activity and poor intestinal absorption, which make it improbable that high concentrations of polyphenols may be found for any length of time in the body after their ingestion if contained within an extract in a“free” form. In a preferred embodiment of the invention, the nanoparticles encapsulate a water-soluble extract of cocoa hulls and/or nibs having a content of polyphenols comprised between 40 and 70 mg/g, preferably between 50 and 70 mg/g, more preferably between 60 and 70 mg/g relative to the total weight of the extract. According to another preferred embodiment of the invention, the nanoparticles encapsulate a water-soluble extract of cocoa hulls and/or nibs comprising polyphenols selected in the group consisting of: /V-caffeoyl aspartate, catechin, epicatechin, quercetin 3-0-glucoside, quercetin 3-0-arabinoside, procyanidin trimer I, procyanidin trimer II, procyanidin trimer III, procyanidin trimer V, procyanidin trimer VI, procyanidin trimer VII, procyanidin dimer I, procyanidin dimer II, procyanidin dimer III and a combination thereof. According to one even more preferred embodiment of the invention, the nanoparticles encapsulate a water-soluble extract of cocoa hulls and/or nibs having a content of polyphenols comprised between 40 and 70 mg/g, preferably between 50 and 70 mg/g, more preferably between 60 and 70 mg/g relative to the total weight of the extract and wherein said polyphenols are selected in the group consisting of: /V-caffeoyl aspartate, catechin, epicatechin, quercetin 3-O-glucoside, quercetin 3-O- arabinoside, procyanidin trimer I, procyanidin trimer II, procyanidin trimer III, procyanidin trimer V, procyanidin trimer VI, procyanidin trimer VII, procyanidin dimer I, procyanidin dimer II, procyanidin dimer III and a combination thereof. In particular, the preferred plant varieties from which it is possible to obtain extracts with the aforesaid characteristics in terms of the total amount and type of polyphenols (taken both individually and in combination) are the Costa Rica, Madagascar and Venezuela Barinas, preferably Costa Rica and Madagascar varieties, even more preferably Costa Rica. Therefore, in one embodiment, the nanoparticles encapsulate a water-soluble extract that is selected from a water-soluble extract of hulls and/or nibs of cocoa of the Costa Rica variety, a water-soluble extract of hulls and/or nibs of cocoa of the Madagascar variety or a water-soluble extract of hulls and/or nibs of cocoa of the Venezuela Barinas variety. In a preferred embodiment of the invention, said water- soluble extract is preferably a water-soluble extract of hulls and/or nibs of cocoa of the Costa Rica variety. In fact, as also disclosed in the section containing example embodiments and comparative examples, the best results as far as dimensions and the polydispersity index are concerned were obtained in the case of nanoparticles encapsulating said water-soluble extract of hulls or nibs of cocoa of the Costa Rica variety. In fact, these nanoparticles have dimensions comprised between 200 and 400 nm, more preferably between 300 and 400 nm, measured by means of the Dynamic Light Scattering (DLS) technique, and a polydispersity index (PDI) comprised between 0.1 and 1.0, preferably between 0.1 and 0.5. According to one embodiment, as regards the efficiency of encapsulation of the extract of cocoa hulls and/or nibs comprising polyphenols, the nanoparticles of the present invention have an encapsulation efficiency of at least 18% by weight, preferably of at least 50% by weight, even more preferably of at least 60% by weight relative to the total weight of the extract to be encapsulated, i.e. the extract used for the preparation of the nanoparticles.

The encapsulation efficiency (EE) is determined by means of the following formula: EE = (M, - M _s )/M, where M is the total mass of extract used for the preparation of the nanoparticles and M _s is the mass found in the supernatant analysed. As also demonstrated in the Examples section, the nanoparticles showing a greater encapsulation efficiency are nanoparticles encapsulating a water-soluble extract with a content of polyphenols comprised between 50 and 70 mg/g, preferably between 60 and 70 mg/g, relative to the total weight of the extract. Said extract is preferably an extract solely of hulls of cocoa of the Costa Rica variety. Therefore, in a preferred embodiment, the nanoparticles of the invention are nanoparticles encapsulating a water-soluble extract of cocoa hulls with a content of polyphenols comprised between 50 and 70 mg/g, preferably between 60 and 70 mg/g relative to the total weight of the extract, preferably an extract of hulls of the Costa Rica variety. Said polyphenols are preferably selected in the group consisting of: /V-caffeoyl aspartate, catechin, epicatechin, quercetin 3- O-glucoside, quercetin 3-O-arabinoside, procyanidin trimer I, procyanidin trimer II, procyanidin trimer III, procyanidin trimer V, procyanidin trimer VI, procyanidin trimer VII, procyanidin dimer I, procyanidin dimer II, procyanidin dimer III and a combination thereof. Said nanoparticles encapsulating said extract show an encapsulation efficiency of at least 60%, preferably comprised between 60 and 70% by weight relative to the total weight of extract to be encapsulated, i.e. the extract used for the preparation of the nanoparticles themselves. The nanoparticles of the present invention can be used in an aqueous suspension or else in a dry form, i.e. in the form of a powder to be reconstituted in an aqueous solvent.

The present invention further relates to the use of said nanoparticles encapsulating an extract of cocoa hulls and/or nibs comprising polyphenols for the preparation of nutraceutical, pharmaceutical, cosmetic or food formulations.

Said formulations are preferably selected in the group consisting of a capsule, tablet, beverage, face cream, body cream, serum or toothpaste. The nanoparticles of the present invention can also be used for the prevention of the cellular oxidative stress, as well as for the treatment or prevention of the risk of pathological conditions of the cardiovascular system of an individual. Said pathological conditions are preferably selected in the group consisting of hypertension, coronary disease, heart attack or dementia.

The present invention also relates to a nutraceutical, pharmaceutical, cosmetic or food composition comprising the nanoparticles according to the present invention, in an aqueous suspension or else in a dry form, i.e. in the form of a powder to be reconstituted in an aqueous solvent, and at least one further excipient ingredient that is acceptable from a nutraceutical, pharmaceutical, cosmetic and/or food point of view. Said at least one further ingredient is preferably selected from among vitamins, mineral salts, carbohydrates, proteins, fats, gelling agents or other excipients such as plasticisers, lubricants, colourings, preservatives and flavourings.

Examples of embodiments and comparative examples

Example 1 - Preparation of nanoparticles encapsulating an extract of cocoa hulls and/or nibs.

1.1 Extraction of polyphenols from raw cocoa hulls and nibs.

The extraction of polyphenolic compounds was carried out following a process based on a known procedure described in the literature (Hammerstone et. al.“High-performance liquid chromatography/mass spectrometry analysis of proanthocyanidins in foods and beverages”, Journal of Agricultural and Food Chemistry, 1999), suitably modified by the Applicant. The cocoa varieties used are the following: Costa Rica, Jamaica, Madagascar, Ecuador bio, Venezuela porcelana, Ghana, Trinidad, Tobago, Grenada and Venezuela Barinas varieties. The raw cocoa hulls and nibs of each of the above-listed varieties were subjected to a grinding step carried out upstream of the extraction process in order to obtain a homogenous starting raw material. In order to obtain maximum efficiency in the extraction of polyphenols during the preparation of samples, the raw material was subjected to the following extraction procedure which comprises the steps of: immersing 15 g of raw material in 125 ml of hexane, mixing the mixture for 20 minutes at 200 rpm, centrifuging for 30 minutes at 4000 rpm and separating the sediment formed from the supernatant, extracting the sediment in 70% acetone in a 1 :5 ratio (i.e. adding 70% acetone to the sediment in an amount that is 5 times the weight of the sediment itself), stirring for 3 minutes and subsequently centrifuging at 4000 rpm for 30 minutes. The extraction procedure was repeated four times on the sediment formed from the supernatant, at the end of which the various supernatants obtained were mixed together and filtered. Finally, the organic solvent was eliminated by evaporation at room temperature and the final extract obtained was lyophilised using a Virtis Advantage-53 instrument (Stereoglass, Perugia, Italy) under the following operating conditions:

Temperature (° C) Minutes

Freezing -35 180

Drying -30 360

-10 360

+10 240

+25 180 The extraction and lyophilisation procedures carried out enabled lyophilised water-soluble, easily manipulate lyophilised extracts to be obtained.

1.2 Determination of the total polyphenol content

The total polyphenol content was determined by following the Folin-Ciocalteu calorimetric method. An aliquot of 50 mI_ of a solution of extract before lyophilisation and after lyophilisation was added to 1.58 ml of water and 100 mI_ of Folin-Ciocalteu reagent.

After 3 minutes, 300 mI_ of sodium carbonate were added and the absorbance was measured by means of a spectrophotometric measurement at 765 nm after 2 hours of shelter from light. The total polyphenol content was then determined by making reference to the calibration curve of gallic acid (r ² = 0.9985) obtained under the same conditions. Similarly, using the same method and the same conditions, the calibration curve of procyanidin B ₃ (r ² =0.9981 ), a dimer of the catechin isolated from plant material by the Applicant, was obtained. The total polyphenol content present in the extracts obtained from the cocoa varieties and the various types of starting material used is shown in the table below (Table 1 ) and is expressed as mg/g of weight of fresh extract.

Table 1

From the data shown in Table 1 , it is possible to observe that the total polypheno content of extracts deriving from hulls versus nibs is not significantly different for a same cocoa variety. It is evident, however, that the content varies a great deal for different cocoa varieties. For this reason, for the purposes of the present invention, only the cocoa extracts containing the largest amount of total polyphenols were selected and used for the subsequent experiments described in Examples 2, 3 and 4. The selected extracts were: extract of hulls of cocoa of the Costa Rica variety, extract of nibs of cocoa of the Costa Rica variety, extract of hulls of cocoa of the Madagascar variety and extract of hulls of cocoa of the Venezuela Barinas variety.

1.3 Chemical characterisation and phenolic profile of the cocoa extracts

The composition of the extracts of hulls and nibs of cocoa of the Costa Rica variety and of hulls of cocoa of the Madagascar variety was determined by FIPLC-PDA/UV-ESI-MS/MS analysis according to the experimental protocol described below. The extracts of hulls of cocoa of the Costa Rica variety, of nibs of cocoa of the Costa Rica variety and of hulls of cocoa of the Madagascar variety were dissolved in MeOH at the concentration of 2.5 mg/ml and centrifuged for 5 minutes at 3200 rpm. The supernatant was analysed by high- performance liquid chromatography (HPLC), coupled with a photodiode array/ultraviolet (PDAJJV) detector and an LCQ Advantage (Thermo Finnigan) electrospray ionisation ion trap mass spectrometer (ESI-MS) equipped with the software application Xcalibur 3.1. The chromatography was performed by injecting a volume of 20 mI_ of the sample into a Luna 5 pm column (C-18, 4.6x150mm, Phenomenex) and eluting with a mixture consisting of MeOH (solvent A) and an aqueous solution of 0.1 % formic acid (solvent B), according to the following linear gradient: 0-50 min; 5-55% A. The elution was performed at a flow of 0.8 ml/min using a 2:8 splitting system, for the MS detector (160 pL/min) and the PDA detector (640 pL/min), respectively. For the MS analysis, use was made of an ESI interface in the negative mode and the following parameters: temperature of the capillary column, 270 °C; voltage of the capillary column, -16.00 V; spray voltage, 4.50 kV; sheath gas (N ₂ ), 60.00 arbitrary units; flow of the auxiliary gas (N ₂ ), 3.00 arbitrary units; collision energy in MS/MS 35%; scanning interval, m/z 150-2000. The PDA data were recorded in an interval with a wavelength comprised between 200 and 600 nm, using three preferential channels at the wavelengths of 254, 280 and 325 nm. As shown in Figure 1 , both extracts of cocoa of the Costa Rica v ariety (deriving from both nibs and hulls, respectively shown in graphs a) and b)) show a similar profile, with a total of 14 phenolic compounds identified, whose numbering, shown below, corresponds to that of the peaks in the figure: a caffeoyl derivative, N-caffeoyl aspartate (1 ); a flavan-3-ol, catechin/epicatechin (9); three procyanidin dimers (5, 6 and 10) and seven trimers (2, 3, 4, 7, 8, 11 and 12); two glycosidic flavonols, quercetin 3-O-glucoside (13) and quercetin 3-O-arabinoside (14).

The extract of hulls of cocoa of the Madagascar variety, by contrast, shows a less rich phenolic profile, above all in terms of procyanidins.

The data related to the peaks, identified with the numbers 1-14, are listed in the table below (Table 2).

All the compounds were identified by comparison of the chromatographic data (retention times, t _Ft ), UV and ESI mass spectrometry data with those previously reported in the literature (Ortega, N. et al., “Comparative study of UPLC-MS/MS and HPLC-MS/MS to determine procyanidins and alkaloids in cocoa samples”. Journal of Food Composition and Analysis, 23(3), 298-305).

Table 2

aThe ions were generated by fragmentation of the deprotonated molecular ion in the ESI- MS/MS experiments and the base peak generated has been highlighted in bold;

bfor compound (9) it was not possible, solely on the basis of the acquired data, to define whether it was catechin or epicatechin, the two compounds being isomeric with each other, with identical UV spectra and molecular ion fragmentation spectra;

cThe MS/MS spectrum was obtained by fragmentation of the adduct [M ⁺ HCOO] .

The analysis of the fragmentation spectra reveals the presence, within the analysed extracts, of ions characteristic of procyanidins. In particular, among the various procyanidins, one observes the presence of ions characteristic of B-type procyanidins, i.e. procyanidins in which the monomeric units (catechin and/or epicatechin) are joined by C ₄ -C ₈ or C ₄ -C ₆ bonds.

1.4 Preparation of the Chitosan HCI FG90 solution

2 g of food grade chitosan (Ch-HCI FG90, Giusto Faravelli, Italy) were suspended in 200 ml of water and added to 7.3 ml of 1 N HCI. The solution was kept under magnetic stirring for 12- 16 hours, filtered and, finally, lyophilised by means of the Virtis Advantage-53 instrument (Stereoglass, Perugia, Italy) under the following operating conditions: Temperature (° C) Minutes

Freezing -35 180

Drying -30 360

-10 360

+10 240

+25 180

1.5 Preparation of the nanoparticles encapsulating the extract

The chitosan (Ch-HCI FG90)-based nanoparticles (NPs) containing the cocoa extract were prepared by ionotropic cross-linking with the cocoa extract itself. In particular, aliquots of 100 mI_ of a solution of extract (22 mg/ml) in water were added to 5 ml of a solution of Ch-HCI FG90 in water (0.25 mg/ml), for a total of 900 mI_, under continuous stirring at room temperature, until obtaining a suspension of nanoparticles.

1.6 Preparation of nanoparticles encapsulating procvanidin B ₃

For comparative purposes, similarly to what was done in Example 1 , paragraph 1.5, chitosan-based nanoparticles (Ch-HCI FG90) encapsulating a solution of procyanidin B ₃ (indicated, for the purposes of the present invention as“procyanidin B ₃ standard”) were prepared. Said nanoparticles were prepared by adding aliquots of 50 mI_ of a solution of procyanidin B ₃ in water (12.5 mg/ml) to 5 ml of a solution of Ch-HCI FG90 in water (0.25 mg/ml), for a total of 150 mI_, under continuous stirring at room temperature until obtaining a suspension of nanoparticles.

1.7 Preparation of the nanoparticles encapsulating the extract bv self-assembly starting from a mixture of powders

The chitosan (Ch-HCI FG90)-based nanoparticles (NPs) containing the cocoa extract were alternatively prepared starting from a mixture of powders comprising commercial ( food grade) chitosan in a powder form (lyophilised) and the water-soluble extract of cocoa in a dry form obtained from the aqueous solution of the extract itself, by lyophilisation. In particular, 1.25 mg of lyophilised Ch-HCI FG90 and 20 mg of lyophilised cocoa extract were dissolved in 5 ml of water, under continuous stirring at room temperature until obtaining a suspension of nanoparticles.

Example 2 - Characterisation of the nanoparticles encapsulating the cocoa extract according to the present invention.

2.1. Dimensional analysis

The nanoparticles produced as described in Example 1 , paragraphs 1 .5 and 1.6 were analysed from a dimensional viewpoint by means of the Dynamic Light Scattering (DLS) technique in order to obtain the average diameter dimensions and the polydispersity index (PI) of the nanoparticles._Figures 2, 3, 4 and 5 show the dimensional analyses of the nanoparticles encapsulating an extract of hulls of cocoa of the Venezuela Barinas variety, an extract of hulls of cocoa of the Madagascar variety, an extract of nibs of cocoa of the Costa Rica variety and an extract of hulls of cocoa of the Costa Rica variety, respectively. These tests showed that only in the case of an extract of hulls or nibs of cocoa of the Costa Rica variety, the nanoparticles encapsulating said extract have diameter dimensions that are ideal for the purposes of the present invention, i.e. a diameter dimension comprised in the order of hundreds of nanometres, and with an acceptable polydispersity index (PI).

Table 3

In the case of the extract of hulls of cocoa of the Venezuela Barinas variety or the extract of hulls of cocoa of the Madagascar variety, the nanoparticles encapsulating said extracts in fact had larger dimensions, less favourable for the purposes of the present invention (i.e. dimensions in the order of pm). This behaviour is likely to be attributable to the different content of polyphenols present in the extract of hulls of cocoa of the Madagascar variety. In fact, as shown in Example 1 , paragraph 1.3, the extract deriving from hulls of cocoa of the Madagascar variety is less rich than the one deriving from nibs or hulls of cocoa of the Costa Rica variety, both as regards the total amount of polyphenols and the type thereof; in particular, the extract deriving from hulls of cocoa of the Madagascar variety is less rich in terms of procyanidins. As regards, on the other hand, the nanoparticles encapsulating the procyanidin B ₃ standard, it is possible to observe that the dimension of said nanoparticles does not differ significantly from that of those encapsulating an extract of cocoa hulls of nibs of the Costa Rica variety, which demonstrates the fact that the presence of procyanidins in large amounts within the extract effectively enables the formation of nanoparticles with the desired dimension. For this reason, the subsequent analysis on encapsulation efficiency was conducted using only nanoparticles encapsulating an extract of hulls or nibs of cocoa of the Costa Rica variety. As regards the nanoparticles produced as described in Example 1 , paragraph 1 .7, these were analysed from a dimensional viewpoint by means of the Dynamic Light Scattering (DLS) technique in order to obtain the average diameter dimensions and the polydispersity index (PI) of the nanoparticles, for the purpose of evaluating the effectiveness of the process for obtaining said nanoparticles by self-assembly also in the event that said process envisages the step of starting from a mixture of powders. Figure 6 shows, by way of example, the dimensional analysis of the nanoparticles encapsulating an extract of hulls of cocoa of the Costa Rica variety obtained as per Example 1 , paragraph 1 .7.

Table 4

This test revealed that, also in the case of nanoparticles produced from a mixture of powders, as described in Example 1 , paragraph 1 .7, said nanoparticles have diameter dimensions that are ideal for the purposes of the present invention, i.e. a diameter dimension comprised in the order of hundreds of nanometres, and with an acceptable polydispersity index (PI).

2.2 Efficiency of encapsulation (EE) of the extract and procvanidin B ₃ in the nanoparticles

With the aim of evaluating both the amount of extract of hulls or nibs of cocoa of the Costa Rica variety and of procyanidin B ₃ actually encapsulated in the nanoparticles obtained as described in Example 1 , paragraphs 1.5 and 1 .6, the latter were centrifuged at 20000 rpm for 1 hour at a temperature of 4 °C. The supernatant was then analysed following the Folin- Ciocalteu calorimetric method in order to determine the encapsulation efficiency (abbreviated as EE) by means of the following formula:

EE = (M, - M _s )/M,

where M is the total mass of extract used for the preparation of the nanoparticles and M _s is the mass found in the supernatant analysed.

Based on the encapsulation efficiency values shown in the table (Table 5), it can be deduced that the nanoparticles prepared with extract of hulls of cocoa of the Costa Rica variety are capable of encapsulating an amount of extract that is almost three times greater compared to the nanoparticles prepared with the extract of nibs of cocoa of the Costa Rica variety.

Table 5

For this reason, the subsequent experiments described in paragraphs 2.3-2.5 and Examples 3 and 4 were carried out using the preferred embodiment of the present invention, i.e. the nanoparticles encapsulating an extract of hulls of cocoa of the Costa Rica variety.

2.3 Z Potential (ZP)

The nanoparticles according to the preferred embodiment of the present invention, i.e. the nanoparticles encapsulating an extract of hulls of cocoa of the Costa Rica variety produced as described in Example 1 , paragraph 1 .5 and the nanoparticles encapsulating the procyanidin B ₃ standard obtained as described in Example 1 , paragraph 1 .6, were analysed by light-scattering (Zetasizer Nano ZS, Malvern) in order to determine the Z Potential values, which are shown in the table below.

Table 4

The nanoparticles prepared as described in Example 1 , paragraph 1.5 were lyophilised and redispersed in a simulated gastric fluid (FGS) consisting of 0.04 M HCI at pH 1.2 rendered isotonic with NaCI (i.e. an aqueous solution consisting of 40 g of 1 N HCI and 1 g of NaCI for 500 ml of solution). Figure 7 shows the dimensional analysis of the nanoparticles encapsulating the extract of hulls of cocoa of the Costa Rica variety, which indicates that said nanoparticles are stable in a simulated gastric fluid (SGF). Their dimension, being in fact equal to 365.1 nm (with a polydispersity index of 0.737), does not differ significantly from that of the same nanoparticles in an aqueous solution. This result shows that the nanoparticles of the present invention are also stable at acidic pH levels and are therefore potentially capable of protecting the extract encapsulated therein from degradation and acid attack in the gastrointestinal tract.

2.5 Morpholoaical characterisation by Scannino Transmission Electron Microscopy (STEM)

The nanoparticles encapsulating an extract of hulls of cocoa of the Costa Rica variety prepared as described in Example 1 , paragraph 1 .5 and encapsulating a solution of procyanidin B ₃ (procyanidin B ₃ standard) as described in paragraph 1 .6 were purified by centrifugation at 20000 rpm for 1 hour at a temperature of 4 °C. The nanoparticles were then diluted 1/200 in ethanol and placed on graphite grids for analysis by means of an FEI Quanta 450 ESEM with a field emission gun (FEG). Figure 8 shows two comparative STEM images illustrating nanoparticles encapsulating an extract of hulls of cocoa of the Costa Rica variety (image a)) and nanoparticles encapsulating the procyanidin B ₃ standard (image b)). It is possible to observe that the nanoparticles have a similar appearance and there are no regions with a different density. Similarly to what was shown by the dimensional analysis in an aqueous dispersion (paragraph 2.1 ), the nanoparticles encapsulating the procyanidin B ₃ standard have a smaller diameter than the particles encapsulating the extract of hulls of cocoa of the Costa Rica variety.

Example 3 - Study on permeation of the cocoa free extract or the free procyanidin B ₃ standard throuah isolated rat intestine, compared with the permeation of the nanoparticles encapsulatina the cocoa extract or encapsulatina the procyanidin B ₃ standard prepared accordina to the present invention

3.1 Materials and methods

For the permeation experiments, nanoparticles encapsulating an extract of hulls of cocoa of the Costa Rica variety according to the present invention were tested and compared with the results obtained for nanoparticles encapsulating the procyanidin B ₃ standard. The results of these experiments were then compared with those obtained with the same extract of hulls of cocoa of the Costa Rica variety in the form of a free extract and with the same procyanidin B ₃ standard, likewise in a free form.

Male Wistar rats with a weight comprised between 250 and 300 g were selected for the experiments and the intestinal mucosa was isolated by means of the following procedure. After each rat was sacrificed, the first 20 cm of the jejunum were immediately removed. The isolated intestine was then cut into strips measuring 1 .5 cm each, the content of the lumen was removed and mounted in an Ussing type chamber (0.78 cm ² of exposed surface), care being taken not to remove the underlying muscle layer. Subsequently, 1 ml of phosphate buffer (pH 6.8 0.13M) rendered isotonic by adding NaCI (phosphate-buffered saline (PBS) 6.8) was added on the apical side, whereas 3 ml of an isotonic buffer solution (pH 7.4, 0.13 M, phosphate buffer (PB) 7.4) were added on the basolateral side (acceptor medium). In order to assure correct oxygenation and stirring of each compartment of the Ussing chamber, a gaseous mixture containing 95% O2 and 5% CO2 was bubbled into each chamber. The Ussing chambers were then positioned in a water bath at a temperature of 37 °C and, after about 20 minutes of equilibration, the medium present in each donor compartment was replaced with 1 ml of a solution of cocoa free extract or 1 ml of a solution of procyanidin B ₃ (procyanidin B ₃ standard) or 1 ml of the suspension of nanoparticles encapsulating the cocoa extract or 1 ml of the suspension of nanoparticles encapsulating the procyanidin B ₃ standard. At 30-minute intervals, for a total of 240 minutes, 1 ml of the sample was drawn from the acceptor compartment and replaced each time with the same volume of fresh medium. The amount of cocoa extract permeated at the various time intervals was then determined with a UV spectrophotometer by means of the Folin-Ciocalteu colorimetric method.

3.2 Results

The study on isolated rat intestine enabled a determination of the rate of permeation of the free extract of cocoa hulls through the isolated rat intestinal tissue compared with the rate of permeation of the extract of cocoa hulls encapsulated in the nanoparticles of the present invention. Based on figure 9 and the table below (Table 5), which shows the data relating to permeation, through the isolated rat intestine, in the case of the free cocoa extract or in the case of a solution of free procyanidin B ₃ or of nanoparticles encapsulating said extract or in the case of a solution of procyanidin B ₃ , after a delay time of about 60 minutes, the permeation rate of the nanoparticles of the present invention is about double that of the extract in free form (permeation promotion factor of 1.9).

Table 5

^* P<0.01

Since the polyphenol concentration in the donor phase is equal to 4 mg/ml in both cases, these results clearly indicate a greater ability (about the double) of the nanoparticles of the present invention to pass through the intestinal barrier and to transport the polyphenols contained therein, compared to the same cocoa extract in free form. Example 4 - Test on cell viabilitv and production of reactive oxyaen species (ROS)

4.1 Materials and Methods

For the experiments on cell viability and the production of reactive oxygen species (ROS), nanoparticles encapsulating an extract of hulls of cocoa of the Costa Rica variety according to the present invention were tested. The results of these experiments were then compared with those obtained with the same extract of hulls of cocoa of the Costa Rica variety in the form of a free extract.

4.2 Isolation of human umbilical vein endothelial cells (HUVECs)

The umbilical cords of normal foetuses were pre-washed in the delivery room under sterile conditions and stored at 4 °C in receptacles containing saline solution, penicillin 50 Ul/ml and streptomycin 50 pg/ml, and used within 24 hours after delivery. The procedure for isolating the cells was carried out under a laminar flow hood in conditions of sterility. The umbilical vein was cannulated with a cannula connected to a syringe, by means of which the saline solution was injected with the aim of freeing the vein of blood and any clots obstructing the lumen of the vessel. After washing, the vein was incubated with collagenase for 10 minutes at a temperature of 37 °C to favour detachment of endothelial cells. The collagenase was then blocked by injecting the vein with Medium 199 (M199) containing 20% foetal bovine serum (FBS). The whole sample was collected in a 50 ml test tube and, after centrifugation, the supernatant was discarded and the residual precipitate was then resuspended in the FIUVEC culture medium: M199 containing FBS (20%), heparin, penicillin, streptomycin, FIEPES, glutamine and bovine retina growth extract. The cell suspension thus obtained was then plated in flasks for cell cultures coated with gelatine (1 %). After 24 hours, the medium was changed after washing with saline solution to eliminate the red blood cells. On reaching confluence, the cells were detached with trypsin and used for the subsequent analyses.

4.3 Treatment of FIUVECs

15000 FIUVECs at passage P3-P4 were seeded in every well of a 96-well multiwell plate in 100 mI of culture medium. The day after seeding, in every well, the culture medium was replaced with the medium M199 containing FBS (5%) at different polyphenol concentrations (5, 10, 25, 50, 100, 250 pg/ml) contained in the cocoa free extract and in the nanoparticles encapsulating said cocoa extract. The cells without the cocoa free extract and without nanoparticles encapsulating said cocoa extract were used as the control. After 2 hours and 24 hours of incubation at 37 °C in a humidified atmosphere containing CO2 (5%), a cell viability test as described in paragraph 4.4 was performed on the cells. After the 2- and 24- hour treatments with the cocoa free extract or with nanoparticles encapsulating said cocoa extract, the cells were incubated with hydrogen peroxide (H2O2, 100 mM) for one hour at 37 °C in a humidified atmosphere containing CO2 (5%) to induce oxidative stress. The cells were then analysed in order to evaluate the production of reactive oxygen species (ROS) (see paragraph 4.5).

4.4 Cell viability test

Cell viability was evaluated through the WST-1 Assay (Roche Applied Science, Mannheim, Germany). In order to evaluate cell viability at the end of the treatment, the cells were incubated with 10 mI of WST-1 and 100 mI of M199 containing FBS (5%) for 3 hours at 37 °C in a humidified atmosphere containing CO2 (5%). The formazan produced was quantified by measuring absorbance at 450 nm in reference to a wavelength of 650 nm with a reader for 96-well multiwell plates (Thermo Scientific Multiskan FC Microplate Photometer).

4.4.1 Results

From the data shown in Figure 10, it is possible to observe that after 2 hours of treatment of the HUVECs with the cocoa extract in the form of a free extract (indicated in the figure as “COCOA”) and with the nanoparticles of the present invention encapsulating said cocoa extract (indicated in the figure as“COCOA NPs”), at the same polyphenol concentrations (10, 25, 50 and 100 pg/ml), there are no significant differences versus the control (untreated HUVECs). Moreover, a comparison of the two treatments (COCOA vs. COCOA NPS) does not reveal any significant difference in cell viability, which always remains very high, i.e. comparable to that of the control (100%), a sign that both the free extract and the nanoparticles encapsulating said extract are nontoxic in a wide range of concentrations. As regards cell viability at 24 hours of treatment, the data shown in Figure 1 1 reveal a significant increase in cell viability at all tested polyphenol concentrations with both types of treatment (COCOA vs. COCOA NPS).

4.5 Test on the production of reactive oxygen species (ROS)

The production of reactive oxygen species (ROS) was evaluated by means of a fluorescent probe 5-(and 6)-chloromethyl-20,70-dichloro-dihydro-fluorescein diacetate, acetyl ester (CM- H2DCFDA).

The HUVECs were incubated with hydrogen peroxide or with M199 containing FBS (5%) for 30 minutes, during which 100 mI of CM-H2DCFDA (10mM) in PBS containing DMSO (9.1 %) were added to each well. The production of ROS was then evaluated by measuring the increase in fluorescence (488 nm, excitation wavelength at 510 nm, emission wavelength) by means of a reader for 96-well multiwell plates (Thermo Scientific Fluoroskan Ascent Microplate Fluorometer).

4.5.1 Results All the analyses relating to the basal production of ROS refer to 100% of the ROS production of HUVECs incubated with hydrogen peroxide (H2O2, 100 mM) and the results are shown in Figure 12. As may be noted, there is no significant difference in the basal production of ROS in the case of untreated HUVECs (CN) and HUVECs treated for 2 hours (Figure 12 A) and 24 hours (Figure 12 B) with cocoa free extract (COCOA) and nanoparticles encapsulating said extract (COCOA NPs) at different polyphenol concentrations (5, 10, 25, 50, 100 and 250 pg/ml). A comparison of the two treatments (COCOA vs. COCOA NPs) likewise does not reveal any significant difference in the production of ROS. In contrast, the reduction in the percentage of ROS in the control (CN) versus cells treated with H2O2 is significant. As shown in Figure 13, in fact, under conditions of stress with hydrogen peroxide, it is possible to observe a significant percentage reduction in ROS in the control versus cells subjected to oxidative stress with hydrogen peroxide. After two hours of treatment one observes a significant reduction in the ROS production of HUVECs treated with the cocoa free extract (COCOA) at all polyphenol concentrations with the exception of the concentration of 5 pg/ml, whilst in the case of the treatment with the nanoparticles encapsulating the extract (COCOA NPS), the reduction is significant only at the polyphenol concentration of 10 pg/ml. In any case, after two hours of treatment, no significant difference is observed between the two treatments (COCOA vs. COCOA NPs) at each tested concentration.

After 24 hours of treatment, by contrast, there is a significant percentage reduction in ROS at all concentrations of cocoa free extract and nanoparticles encapsulating said extract with the exception of the polyphenol concentration of 5 pg/ml in the case of nanoparticles. These data demonstrate that the cocoa extracts with a high content of polyphenols in the form of free extracts have the ability to protect cells against oxidative stress and that the extracts encapsulated within the nanoparticles of the present invention show the same ability.