Nanoencapsulation of olive phenolic compounds in protein matrices

Abstract

A formulation for nutraceutical and / or pharmaceutical use based on protein nanoparticles is described, including a bioactive compound chosen from hydroxytyrosol, tyrosol, olive phenolic compounds, and mixtures thereof, and a zein protein matrix stabilized with sodium deoxycholate.

Description

[0001] "NANOENCAPSULATION OF OLIVE PHENOLIC COMPOUNDS IN

[0002] PROTEIN MATRICES"

[0003] * * *

[0004] TECHNICAL FIELD OF THE INVENTION

[0005] 5 The present invention relates to the nano-encapsulation of phenolic compounds of the olive tree in protein matrices .

[0006] The present invention also relates to a formulation for nutraceutical or pharmaceutical use based on phenolic compounds of the olive tree, nano-encapsulated in protein

[0007] 10 matrices .

[0008] In particular, the present invention refers to polymeric nanoparticles , preferably zein-based, for the del ivery of phenolic compounds of the olive tree : hydroxytyrosol , tyrosol and derivatives thereof .

[0009] 15 KNOWN ART

[0010] As is well known, the Mediterranean diet is considered to promote numerous advantages for human health, such as a low incidence of chronic and degenerative diseases , and these particular benef its have been attributed to extra virgin

[0011] 20 olive oil (EVO) by many studies , including Willet et al . ,

[0012] 1995 ; Tripoli et al . , 2005 ; Huang et al . , 2008 . The beneficial ef fects of EVO depend on an adequate composition of fatty acids , characteri zed by a high percentage of monounsaturated derivatives (MUFAs ) , but above all on

[0013] 25 phenolic secondary metabolites that characteri ze it compared to other types of seed oils rich in MUFAs , but without the same beneficial ef fects . Furthermore , unli ke most edible oil s , EVO is obtained from the sole pressing of the fruits of the olive tree ( Ol ea europaea L . ) which have not undergone

[0014] 1 treatments other than washing, centri fugation, filtration and decantation, thus preserving all the beneficial properties typical of its minor components . These include lipophilic and hydrophilic compounds that act as natural

[0015] 5 antioxidants , playing an inhibitory role in lipid peroxidation of the foods wherein they are contained . This means that they have a double protective action, first on the oil and then on the man who consumes it (Visioli et al . ,

[0016] 2011 . The antioxidant activity of phenolic compounds present

[0017] 10 in food can be explained by the so-called polar paradox : in a bulk oil system, antioxidants such as phenolic secoiridoids from the olive tree are oriented at the air-oil interface , thus protecting the lipophilic molecules contained in them from oxidation . Thus , these compounds improve the resistance

[0018] 15 of ol ive oil to oxidation, give it pecul iar taste characteri stics and have a positive ef fect on human health .

[0019] These phenolic secoiridoids are contained in the unsaponi f iable fraction of EVO, which constitutes about 2 % of its total weight and is also made up of a series of

[0020] 20 heterogeneous compounds such as aliphatic and triterpene alcohols , sterols , hydrocarbons , volatile compounds and antioxidants ( carotenes , tocopherols and phenols . The most abundant phenol ic secoiridoids in olive fruit are oleuropein, demethyloleuropein and ligstroside which, during

[0021] 25 mechanical extraction and oil storage , are hydrolysed by endogenous p -glucosidases giving rise to aglyconic derivatives (Bulotta et al . , 2013 ; Bremes et al . , 2001 ) .

[0022] Thus , the most abundant phenolic secoiridoids in oi l are oleacein ( 3 , 4 -DHPEA-EDA) and oleocanthal (p-HPEA-EDA) which

[0023] 30 represent the dialdehyde form of decarboxymethyl helenol acid bound to hydroxytyrosol or tyrosol respectively and oleuropein and ligstroside aglycones , known as 3 , 4 -DHPEA-EA

[0024] 2 and p-HPEA-EA, respectively, as well as the simple phenolic alcohols 3, 4-dihydroxyphenylethanol , also known as hydroxy tyrosol (3, 4-DHPEA or HTyr) , and P- hydroxyphenylethanol, or tyrosol (p-HPEA or Tyr) (Bremes et

[0025] 5 al . , 2001; Boskou 2015; Bendini et al., 2007) .

[0026] In recent years , many biological properties have been attributed to olive phenols, and both total oil / leaf / fruit extracts and isolated compounds have been studied . The effects of phenolic secoiridoids are of particular interest

[0027] 10 in view of their possible pharmacological application as purified and neo-synthesized compounds in single form or as an active component in the enrichment of food matrices. The benefits of olive secoiridoids have been well documented and include cardioprotective, antitumor, antimicrobial ,

[0028] 15 antiplatelet, antiatherogenic and neuroprotective effects

[0029] (Visioli et al., 2011; Hamdi et al., 2005; Bisignano et al.,

[0030] 1999; Petroni et al., 1995; Bazoti et al . , 2006; Sus al it et al . , 2011; Sindona et al . , 2012; Parzonco et al., 2013) .

[0031] In the context of the health effects of EVO, the

[0032] 20 European Food Safety Authority (EFSA) has recently provided a scientific opinion on the relationship between phenols contained in oil and the protection of LDL particles from oxidative damage (EFSA Journal 2011, 9(4) : 2033) as well as the maintenance of normal concentrations of HDL cholesterol

[0033] 25 in blood (EFSA Journal 2012, 9 (8) : 2848 : "Olive oil polyphenols contribute to the protection of blood lipids from oxidative stress") . This opinion can only be expressed in cases where the olive oil contains at least 5 mg of HTyr and its derivatives (oleuropein complex and tyrosol) per 20

[0034] 30 g of olive oil. Consequently, these beneficial effects would be obtained with the daily intake of 20 g of an olive oil

[0035] 3 that has a concentration of at least 250 mg / kg of HTyr and its derivatives.

[0036] In addition, more recently, EFSA has authorised the placing on the market of hydroxytyrosol as a novel food

[0037] 5 ingredient for the general population, excluding children under three years of age and pregnant and lactating women, up to a maximum of 0.215 g / kg in fish and vegetable oils

[0038] (excluding olive oils and olive pomace oils) , and a maximum of 0.175 g / Kg in spreadable fats, excluding their use for

[0039] 10 cooking, baking and frying (EFSA Journal 2017; 15(3) :4728) .

[0040] However, hydroxytyrosol (HTyr) is a compound that is unstable to light and prone to easy degradation, characteristics that compromise its stability during shelf-life.

[0041] For this reason, it has become necessary to develop

[0042] 15 technologically advanced formulations capable not only of protecting its stability over time, but also of optimizing its absorption in the intestine, thus improving its overall effectiveness in the body.

[0043] In particular, HTyr has been described to be extensively

[0044] 20 metabolized in the intestine and liver, undergoing dosedependent absorption in the intestine. In intestinal sorbent cells, also known as enterocytes , HTyr is hydrolysed and subsequently transformed into glucuronide, methylated, and sulphated byproducts. When administered in olive oil, most

[0045] 25 of the compound is detected in plasma and urine in the form of glucuronide . Only 2% of the unmetabolized form is detectable in these fluids (Miro-Casas et al., 2003; Suarez et al . , 2011) . However, the bioactive compound has a high hydrophilicity which is the main barrier to its potential

[0046] 30 application in food.

[0047] 4 Currently, various formulations containing HTyr are already on the market as supplements , for instance

[0048] Oleaselect™ ( Indena, Mi lan, Italy) , based on extracts rich in HTyr or Micotirosolo (NutraLabs , Modena, I taly) , or

[0049] 5 Hydroxytyrosol plus+ ( Granatum, Murcia, Spain) .

[0050] In addition, several delivery systems have been proposed for the delivery of this compound such as macro emuls ions , multiple emulsions , liposomes and lipid sol id particles .

[0051] 10 However, the use of naturally occurring polymers , such as proteins , has steadily increased in several areas of research . In many cases , their use is mainly due to their biocompatibility, biodegradability and formulation versatility, which allow them to be applied for nutraceutical

[0052] 15 or biomedical purposes .

[0053] In fact , formulations obtained from protein polymers have numerous advantages over systems consisting of synthetic derivatives , such as marked biodegradabi lity, better commercial availability and a high ability to interact

[0054] 20 with the encapsulated compound . This occurs by virtue of the dif ferent functional groups present in the structure o f the proteins that promote their interaction with both hydrophilic and lipophilic compounds , and that facil itate the modulation of the surface architecture of the obtained

[0055] 25 systems without the need for any synthetic modi fication .

[0056] Some proteins , in particular, have the status of

[0057] "Generally Recogni zed as Safe" ( GRAS ) which could greatly facilitate the clinical translation of the formulation obtained . In addition, the possibility of developing

[0058] 30 nanoparticle systems us ing simple methods using non-toxic

[0059] 5 solvents is an advantage related to the use of these polymers .

[0060] In this regard, zein, the main storage protein contained in the endosperm of maize, represents the polymer selected

[0061] 5 to obtain particulate systems useful f or biomedical- pharmaceutical and food purposes . In 1985, zein received

[0062] GRAS status from the U.S. Food and Drug Administration (FDA) as a suitable material for use in the coating of pharmaceutical products (Zhang et al., 2015) . On the basis

[0063] 10 of solubility and sequence homology, zein can be classified into : a-zein (characterized by an average molecular weight between 19-22 KDa) , 0-zein (14 KDa) , y-zein (between 16-27

[0064] KDa) and 6-zein (10 KDa) (Anderson et al., 2011) .

[0065] The poor physical stability of pH-neutral zein

[0066] 15 nanoparticles , even after the freeze-drying process, has hindered their application in the food and pharmaceutical fields, probably due to the isoelectric point of the protein itself (Ip ~ 6.2) . For this reason, several stabilizers such as surfactants or other globular proteins have been studied

[0067] 20 in the literature in order to modulate the colloidal interactions of nanoparticles (Zhang et al., 2014) .

[0068] Commercially, zein is available in two forms : yellow and white. The former has a purity of around 88-90% and contains a high percentage of xanthophylls, i.e. pigments

[0069] 25 consisting of lutein, zeaxanthin and p- cryptoxanthin

[0070] (Paliwal et al . , 2014) . White zein, with a purity around

[0071] 98%, is obtained by discoloration of yellow zein and has a negligible content of xanthophylls (Paliwal et al., 2014) .

[0072] Recently, zein has been identified as a protective

[0073] 30 material for drugs passing through the gastrointestinal (GI)

[0074] 6 tract , limiting their degradation or promoting their targeted release into colon . In addition, the protein is insoluble in aqueous environments at pH <11 , which allows it to prevent the release of the active ingredient into the

[0075] 5 upper gastrointestinal tract and provide a targeted release of the encapsulated compound into colon ( Paliwal and

[0076] Palakurthi , 2014 ) . In addition, the good mucoadhesiveness of zein allows to implement the absorption of encapsulated bioactive compound molecules in nanoparticle systems

[0077] 10 ( Penalva et al . , 2015 ) .

[0078] When administered orally, protein nanoparticles move slowly along the gastrointestinal tract , interacting with the protective layer of mucus that l ines the epithelium of the small intestine wherein they become trapped . The

[0079] 15 nanosystems showed a long residence time in the intestine , about 24-48 h, before being degraded and / or eliminated . Thi s phenomenon is related to the fact that zein i s relatively res istant to gastric enzymes . Subsequently, the protein is digested by enzymes in the intestinal fluid, resulting in

[0080] 20 the release of the encapsulated compound (Bhushani et al . ,

[0081] 2017 , Hurtado-Lopez and Murdan, 2006 , Penalva et al . , 2015 ) .

[0082] Based on its stability in acidic environments , zein has al so been studied for potential applications in colon, where it is digested by the microbiota ( Tran et al . , 2019, Bisharat

[0083] 25 et al . , 2019 ) .

[0084] Methods are known wherein zein particles combined with hydroxy tyrosol obtained by electrospray technique are described . However, the particles thus obtained are col loidally unstable and not adequately ef fective for

[0085] 30 systemic administration as electro spraying generates non- uni form and poorly porous particles , with a release poorly

[0086] 7 controllable and independent of pH or intestinal enzymes . In addition, exposure to high voltages can compromise the stability of the active ingredient and reduce the functional eff icacy o f the formulation .

[0087] 5 Zein and polysaccharide-based systems are also known .

[0088] Polysaccharides of fer good mechanical protection to the particles thus obtained, but do not actively participate in the intestinal absorption process , making it insuf ficient and scarcely effective .

[0089] 10 There are also known delivery systems wherein zein is replaced by alginates , vegetable gums , whey or other protein components . However, these systems have signif icant formulation and functional limitations . In particular, alginate and vegetable gums form unstable gelatinous

[0090] 15 lattices in an acidic environment (pH < 2 ) , with consequent early release of the active ingredient , and do not show a behaviour sensitive to the presence of gastrointestinal enzymes . Whey proteins , on the other hand, are vulnerable to low-pH denaturation and pepsin action, compromis ing system

[0091] 20 integrity and promoting uncontrol led release of the encapsulated compound . As a result , these matrices do not guarantee a targeted and gradual or pH- / enzyme-dependent release of bioactive compounds , as is the case in the system developed in the present invention based on zein and sodium

[0092] 25 deoxycholate , which has been specifically designed to resist the gastric environment and release the active ingredient selectively in the intestine .

[0093] Unless speci fically excluded in the detailed description below, what is described in thi s chapter is to

[0094] 30 be considered as an integral part of the detailed description of the present invention .

[0095] 8 SUMMARY OF THE INVENTION

[0096] Facing the problems highlighted by the known art , according to the present invention, nanoparticles have been developed, and a formulation that contains them, suitable

[0097] 5 for releasing in a targeted way into the intestine a bioactive compound comprising hydroxytyrosol (HTyr ) , tyrosol

[0098] ( Tyr) and more generally the polyphenols obtainable by extraction from olive flowers / leaves / fruits , improving their absorption and stability in real biological conditions .

[0099] 10 Since bioactive compounds such as hydroxytyrosol , tyrosol and, more generally, the phenolic compounds present in the olive tree in particular the polyphenols of olive oil are chemically unstable and tend to degrade easily, the aim of the invention is to develop a formulation that

[0100] 15 stabili zes them, in order to ensure their structural integrity and promote their systemic absorption, in order to allow the performance o f their biological actions .

[0101] The purpose of the present invention is therefore to provide a formulation based on hydroxytyrosol , tyrosol and

[0102] 20 more generally the phenolic compounds of the ol ive tree obtained from olive leaves and fruits to be used for nutraceutical use , in such a way as to increase the fraction of unaltered HTyr that can reach the systemic circulation, increase its stability and protect it from oxidation, in

[0103] 25 order to enhance its health benef its .

[0104] In addition, due to its antioxidant and antimicrobial properties , the encapsulation of HTyr in biocompatible matrices has been shown to have a positive impact in the food industry as it has preserved its ability to delay lipid

[0105] 30 oxidation, exerting a beneficial ef fect on the shel f life and stabil ity of food products .

[0106] 9 According to the present invention, a formulation for nutraceutical use is made based on hydroxytyrosol, tyrosol and more generally the phenolic compounds extracted from the leaves and fruits of the olive tree nano-encapsulated in a

[0107] 5 protein matrix, as defined in claim 1.

[0108] Another purpose of the invention are formulations for pharmaceutical use intended for the treatment of diseases caused by oxidative damage and which benefit from the administration of effective quantities of these formulations

[0109] 10 and the nanoparticles contained in them.

[0110] These and other purposes , advantages and characteristics of the present invention will be better specified in a detailed description of the preferred embodiments, which follows. In addition, the claims describe

[0111] 15 preferred variants of the invention, forming an integral part of this description.

[0112] BRIEF DESCRIPTION OF THE FIGURES

[0113] For a better understanding of the present invention, reference is now made to the attached figures, by way of not

[0114] 20 limiting example, wherein:

[0115] Figure 1 shows the mean size, polydispersion index and zeta potential of zein nanoparticles prepared with

[0116] 2 mg / mL polymer, 1.25% w / v sodium deoxycholate (SD) and different concentrations of hydroxytyrosol (HTyr) ;

[0117] 25 - Figure 2 shows the Turbiscan Stability Index (TSI) of zein formulations (2 mg / mL polymer, 1.25% w / v SD) prepared with 0.2-2 mg / mL HTyr, wherein analyses were performed at 25°C (A) and 37°C (B) for one hour;

[0118] Figure 3 shows the evaluation of the encapsulation

[0119] 30 efficiency (EE%) (A) and loading capacity (LC%) (B) of

[0120] 10 HTyr in zein polymer matrices as a function of the amount of active ingredient used in the preparation phases of the nanosystems, according to the invention;

[0121] Figure 4 shows the FT-IR spectrum of zein, sodium

[0122] 5 deoxycholate (SD) , HTyr, physical mixture of powders, empty and containing the bioactive compoundnanosystems ;

[0123] - Figure 5 shows the release profiles of hydroxytyrosol

[0124] (HTyr) in PBS (0.01 M, pH 7.4, 37°C) (panel A) and in

[0125] SGF (0.32% w / v pepsin, pH 1.2) and SIF (1% w / v

[0126] 10 pancreatin, pH 6.8) (panel B) from zein particles (2 mg / mL polymer, 1.25% w / v SD containing 50-800 pg / mL compound) , according to the invention;

[0127] Figure 6 shows the evaluation of the antioxidant activity of hydroxytyrosol (HTyr) in free form or

[0128] 15 encapsulated in zein particles on CaCo-2 cells as a function of bioactive compound concentration, wherein the data are expressed as a percentage of cell viability obtained by MTT assay, the results were obtained from the average of three different experiments ± standard

[0129] 20 deviation . *p <0.05; **p <0.001 compared to H2O2 2100 pM.

[0130] DETAILED DESCRIPTION OF THE INVENTION

[0131] In this document, the term " about" as used herein when referring to a measurable value such as a quantity, a time

[0132] 25 duration, and the like, is intended to encompass variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value, wherein such variations are appropriate to perform the methods described.

[0133] According to the present invention, the formulation for

[0134] 30 nutraceutical use comprises phenolic compounds from the

[0135] 11 olive tree, meaning both the total extracts of oil / leaves / fruit and the isolated compounds, in particular hydroxytyrosol, tyrosol and their derivatives, encapsulated in biocompatible protein nanoparticles. The formulation is

[0136] 5 suitable for use as an antioxidant.

[0137] According to one aspect of the invention, the protein matrix is based on zein and the content of hydroxytyrosol, tyrosol and phenolic compounds of the olive tree within the aqueous or organic phases described below is indicated below.

[0138] 10 In particular, zein is made up of different protein fractions

[0139] (blends ) , including : a-zein ( about 80% of the total , characterized by an average molecular weight between 19-22

[0140] KDa) , 0-zein (10-15% of the total, 14 KDa) , y-zein (10-15% of the total, between 16-27 KDa) and 6-zein (about 3% of the

[0141] 15 total , 10 KDa) . Zein is commercially available in two forms: yellow and white, characterized by different purities.

[0142] According to one aspect of the invention, nanoparticles consist of zein nanospheres stabilized with the anionic surfactant sodium deoxycholate, having an average diameter

[0143] 20 between 100 nm and 200 nm, preferably between 140 nm and 160 nm and even more preferably equal to about 150 nm.

[0144] According to the present invention, zein nanoparticles for the delivery of hydroxytyrosol, tyrosol and phenolic compounds from olive trees are obtained by the

[0145] 25 nanoprecipitation process of the natural polymer zein preformed in aqueous solution as described below.

[0146] Usable hydroxytyrosol (hereinafter also HTyr) has CAS

[0147] Number 10597-60-1.

[0148] Usable tyrosol (hereinafter also Tyr) has CAS Number

[0149] 30 501-94-0.

[0150] 12 Usable Oleacein has CAS Number 149183-75-5.

[0151] Usable oleuropein aglycone has CAS Number 31773-95-2.

[0152] Olive phenolic compounds can be obtained as described in Oliverio et al. (Oliverio M. , Nardi M . , Di Gioia M.L.,

[0153] 5 Costanzo P. , Bonacci S . , Mancuso S . , Procopio A. Semisynthesis as a tool for broadening the health applications of bioactive compound olive secoiridoids: a critical review.

[0154] Natural Product Reports , 2021, DOI : 10.1039 / d0np00084a) .

[0155] Below are the representative structures of the main olive

[0156] 10 phenols .

[0157] These include oleuropein and ligstroside, which are the two main secoiridoid glycosides found in drupes. The combined action of endogenous p-glucosidases and methylesterase and

[0158] 15 the chemical reactions that occur at different stages of EVO production, transform them into the aglyconic derivatives, respectively oleuropein aglycone (3, 4-DHPEA-EA) and ligstroside aglycone (p-HPEA-EA) and their

[0159] 13 decarboximethylated analogues 3 , 4-DHPEA-EDA and p-HPEA-EDA, usually referred to as oleacein and oleocanthal , respectively. Also noteworthy are verbascoside, nuzhenide and nuzhenide oleoside, which have tyrosol and

[0160] 5 hydroxytyrosol in their structure ( Frisina et al . , 2023,

[0161] Oliverio et al . , 2021) .

[0162] The usable zein has CAS Number 9010-66-6.

[0163] The usable sodium deoxycholate monohydrate (hereinafter also SD) has CAS Number 145224-92-6.

[0164] 10 The process of preparing the nanoparticles of the invention comprises the following fundamental steps. i) Preparing an organic phase at room temperature wherein an aliquot of zein 1.5-2.5 mg / ml is added to a hydroalcoholic solution, preferably an ethanol / water

[0165] 15 solution (2:1 v / v) ;

[0166] ID Preparing an aqueous solution of sodium deoxycholate monohydrate at room temperature, preferably at a concentration of 1-2% w / v; ill) Adding a bioactive compound chosen from

[0167] 20 hydroxytyrosol, tyrosol, phenolic compounds from olive trees and mixtures thereof, depending on their lipophilic or hydrophilic chemical nature, i . e . a hydrophilic compound such as hydroxy tyrosol can be added indifferently to the organic phase prepared in

[0168] 25 step i) or to the hydrophilic solution of step ii) , while a lipophilic compound will necessarily be added to the organic phase of step i) , the expert in the field will be able to choose the most suitable addition method for the success of the process; in this way there

[0169] 30 can be two different types of mixtures, a mixture i ) -A and a mixture ii)-B;

[0170] 14 iv) Mixing the organic phase of step i) with mixture ii)-B or mix mixture i)-A with the solution of Sodium deoxycholate monohydrate prepared in step iii) in a ratio of 1-3:5-10 (v / v) , respectively;

[0171] 5 v) Evaporating the alcohol present in the mixture of step iv) possibly operating under vacuum, aatt a temperature below 37 °C and under stirring; in this way the formation of nanoparticles according to the invention takes place ;

[0172] 10 vi) Purifying, possibly by centrifugation, eliminating the supernatant and the unreacted compounds and subsequently, where appropriate, concentrating or freeze-drying and resuspending / reconstituting the freeze-dried in water.

[0173] 15 With reference to step iv) the ratio 1-3 : 5-10 (v / v) will be understood as follows:

[0174] 1-3:5-10 (v / v) for mixture [i) ] : [ii) -B]

[0175] 1-3:5-10 (v / v) for mixture [i) -A] : [ ii ) ]

[0176] The final product can be marketed as a freeze-dried

[0177] 20 powder to be reconstituted or as an aqueous suspension. The nanoparticles thus obtained can be taken orally with the great advantage of reaching the intestine without undergoing substantial degradation in the gastric environment.

[0178] The inclusion of sodium deoxycholate monohydrate had a

[0179] 25 decisive impact on the functionality of zein nanoparticles.

[0180] In its absence, the latter would have been highly unstable in the gastric environment; in fact acidity causes a rapid breakdown of the protein matrix, with premature release of the active ingredient and a drastic reduction in

[0181] 30 b i o a va i 1 ab i 1 i t y .

[0182] 15 It was experimentally found that the effect of sodium deoxycholate, initially not expected in these terms , was dual and synergistic: on the one hand, it stabilized the nanoparticles at acidic pH thanks to electrostatic and

[0183] 5 hydrophobic interactions with zein, on the other hand, it promoted a more controlled and targeted release in the intestine . This result not only significantly improved the performance of the formulation but also opened new perspectives on the use of bile salts as intelligent

[0184] 10 modulators of oral release, far beyond their traditional role as solubilizers. The observed behaviour suggests that, at least in the case of sodium deoxycholate, bile salts may play an active role in modulating oral release, beyond their conventional function as solubilizing agents . Further

[0185] 15 studies will be needed to verify whether similar effects can also be obtained with other bile salts.

[0186] In practice, as can be seen from the graphs in figure

[0187] 5, the nanoparticles of the invention show a percentage of release of the encapsulated bioactive compound less than

[0188] 20 15%, preferably <12%, more preferably between 7% and 12%, after 2 hours of incubation at pH 1.2. In a slightly different way, in the intestinal environment, the bioactive compound resulted in a more gradual and controlled release; however, after 6 hours the nanoparticles showed a percentage

[0189] 25 of intestinal release greater than 48%, preferably in the order of 70%, 60%, 51% and 48%, depending on the concentration of the bioactive compound, confirming an inverse relationship between the degree of encapsulation and the percentage of intestinal release of the bioactive

[0190] 30 compound .

[0191] 16 The following examples are provided to il lustrate the invention and are not to be considered limiting its scope .

[0192] EXAMPLES

[0193] The applicant has carried out experimental trials , some

[0194] 5 of the results of which are shown in the figures , to support the ef ficacy o f the bio compatible protein matrix to encapsulate hydroxytyrosol , tyrosol and phenolic compounds from olive tree .

[0195] In particular, the following materials were used in the

[0196] 10 trial conducted by the Applicant : zein, sodium deoxycholate monohydrate ( SD) , pepsin derived from porcine gastric mucosa

[0197] ( >400 units / mg protein) , pancreatin obtained from the porcine pancreas ( >3 x USP speci fications ) , tetrazolium salts

[0198] (used for the MTT test ) , saline phosphate buffer ( PBS )

[0199] 15 tablets , dimethyl sul foxide, amphotericin B solution ( 250 pg / ml ) ; they were purchased from Sigma Aldrich (Milan,

[0200] Italy) .

[0201] Hydroxytyro sol was prepared in the Green Chemi stry laboratory of the Magna Graecia University of Catanzaro from

[0202] 20 plant matrices (vegetation water, olive leaves , etc . ) .

[0203] PREPARATION OF PHENOLIC COMPOUNDS

[0204] Hydroxytyrosol can be obtained by synthesi s as already reported by Procopio et al . ( J . Agric . Food Chem. 2009 , 57 ,

[0205] 11161- 11167 , DOI : 10 . 1021 / j f 9033305 ) . In detail , the

[0206] 25 synthesis is obtained from 3 , 4-hydroxyphenylacetic acid and ethanol in the presence of 10% H2SO4 . The ethyl ester obtained is reduced to hydroxytyrosol with NaBH4 .

[0207] HTyr can be obtained by acid hydrolysis of oleuropein, of which the biomass of olive processing is rich, conducted

[0208] 17 in an HC1 solution (15 mM) at room temperature for 48 hours

[0209] (Natural Product Reports, 2021, DOI: 10.1039 / d0np00084a) .

[0210] For the in vitro studies on the release of the active ingredients contained in zein nanoparticles, the following

[0211] 5 were used : DMEM culture medium, enriched with Glutamax I, trypsin / EDTA, penicillin / streptomycin solution and fetal bovine serum (FBS) provided by GIBCO (Life Technologies,

[0212] Monza, Italy) . The human cell line of colon cancer cells

[0213] (CaCo-2) was purchased from ATCC (American Type Culture

[0214] 10 Collection) code HTB-37.

[0215] Zein nanoparticles were obtained by nanoprecipitation of the preformed polymer in aqueous solution as follows.

[0216] PREPARATION OF ZEIN NANOPARTICLES CONTAINING HTyr / Tyr

[0217] Zein (CAS Number 9010-66-6, 25-40 KDa) was solubilized

[0218] 15 at room temperature in an ethanol / water mixture (3-30 ml) with a 2 : 1 ratio (v / v) . Variable amounts of hydroxytyrosol

[0219] (HTyr) 0.2-2 mg / mL were added to this solution, corresponding to 0.1-1 mg of bioactive compound per mg of zein, previously solubilized in the aqueous component of the organic mixture,

[0220] 20 until complete solubilization in the organic polymer phase.

[0221] In parallel, the aqueous phase, (5-50 mL ) containing sodium deoxycholate monohydrate (SD) as an anionic surfactant, was prepared in concentrations ranging from 0.5% to 2% (w / v) . However, the volume of the aqueous phase was

[0222] 25 chosen so that, at the end of the evaporation of the organic solvent, the final concentration of zein in the dispersion was between 1.5 and 2.5 mg / mL.

[0223] The organic phase containing zein and bioactive compound was slowly added (dropwise) to the aqueous phase

[0224] 30 under constant magnetic stirring (600 rpm) .

[0225] 18 The ratio between the organic and aqueous phases was maintained in the range of 3 : 5 (v / v) , depending on the formulation system to be obtained.

[0226] The stirring continued for 12 hours at room temperature,

[0227] 5 favouring both the self-assembly of the nanoparticles and the evaporation of the organic solvent (ethanol) . The nanoparticles were purified of the non-encapsulated active substance and other compounds not integrated into the colloidal structure by ultracentrifugation at 90000 x g for

[0228] 10 60 minutes at 4°C. The centrifuge used was an Optima MAX-

[0229] XP.

[0230] Zein nanoparticles containing variable amounts of tyrosol (Tyr) , given the hydrophilic nature of the compound, were prepared in a way similar to the procedure described

[0231] 15 above .

[0232] PREPARATION OF ZEIN NANOPARTICLES CONTAINING PHENOLIC

[0233] COMPOUNDS

[0234] The preparation of zein nanoparticles containing lipophilic phenolic compounds, such as oleacein or aglycone,

[0235] 20 involved a slight change in the order of addition of the components compared to the procedure previously described.

[0236] In particular, the lipophilic compound was initially solubilized in the organic phase consisting mainly of ethanol . Subsequently, the amount of water necessary to

[0237] 25 obtain the ethanol / water mixture in the ratio of 2: 1 (v / v) was added, followed by zein, until the polymer matrix was completely dissolved.

[0238] The aqueous phase was prepared separately, as in the standard method, using a volume of 5 to 50 mL of water,

[0239] 30 containing sodium deoxycholate monohydrate (SD) as an

[0240] 19 anionic surfactant, in concentrations ranging from 0.5% to

[0241] 2% (w / v) .

[0242] The two phases were subsequently mixed by drop-by-drop addition of the organic phase into the aqueous phase under

[0243] 5 magnetic stirring (600 rpm) , followed by evaporation of the organic solvent and spontaneous formation of the nanoparticles. All operating conditions were kept identical to those used in the formulation with hydrophilic compounds.

[0244] PARTICLES CHARACTERIZATION

[0245] 10 The size, size distribution, and surface charge of the nanoparticles were evaluated by Photon Correlation

[0246] Spectroscopy (PCS) , using a Zetasizer Nano ZS spectrophotometer (Malvern Instruments , UK) . The measurements were conducted in quartz cuvettes at 25°C.

[0247] 15 The optical and physical parameters used for the analysis were: refractive index of the material : 1.59 ( real ) , 0.00

[0248] ( imaginary) ; refractive index of the medium: 1.330;

[0249] 20 • viscosity of the medium: 1.0 mPa s ; dielectric constant: 80.4.

[0250] The instrument was calibrated using a monodisperse standard of polystyrene nanoparticles (126 nm) .

[0251] For each s amp 1 e , three analytical cycles were

[0252] 25 performed, each consisting of ten consecutive measurements.

[0253] The data are reported as an arithmetic mean ± standard deviation .

[0254] The zeta potential was determined with the same instrument by laser Doppler electrophoresis, applying the

[0255] 20 Smoluchowski constant F (K -a) 1.5 for the conversion of electrophoretic mobility.

[0256] The polydispersion index (PDI) represents a descriptive parameter of the particle size distribution; values close to

[0257] 5 zero indicate greater uniformity in the size of nanoparticles .

[0258] Values below 0.2 were observed for the most stable formulations and considered to be an expression of high particle monodispersion.

[0259] 10 Stability studies ( Turbiscan Lab®) were conducted wherein the formulations were placed in cylindrical glass tubes and subjected to stability analysis using the Turbiscan

[0260] Lab® Expert . These measurements allow for early identification of phenomena such as sedimentation,

[0261] 15 flocculation or creaming, even before they are visible to the naked eye, providing a reliable assessment of overall colloidal stability. The stability analysis of the nanoparticles was carried out by evaluating the variation of the backscattering profile (ABS) and transmittance (AT) .

[0262] 20 The equation applied was as follows: wherein: X* is the average speed of free photon transport in the analyzed dispersion.

[0263] From the physical point of view, the X* (<p / d) , estimated

[0264] 25 in the analyzed dispersion, is calculated using the following equation :

[0265] X * (4>, d) = [2d / 3<|)(l — gOQs]

[0266] 21 wherein <p is the volume of the fraction of particles , d is the average diameter of the particles , and g (d) and Qs (d) are optical parameters derived from Mie ' s theory .

[0267] The BS data obtained are then processed as the Turbiscan

[0268] 5 Stability Index ( TS I ) using the TurbiSoft software .

[0269] The measurements were conducted using a pulsating light laser operating in the near infrared at a wavelength of 880 nm for 1 h . Two di fferent optical sensors receive the transmitted and scattered light from the samples at an angle

[0270] 10 o of 180 ° and 45 to the incident light . The two sensors scan the entire height ( 8 -10 cm) of the various samples ( 10 ml ) for 1 h . The experimental data were correlated as a percentage to the luminous flux of two reference standards consisting of a polystyrene latex suspension (no

[0271] 15 transmittance and maximum backscattering ) and silicone oil

[0272] (maximum transmittance and no backscattering) .

[0273] The zein-based nanosys terns , stabili zed with SD and containing increasing concentrations of HTyr, showed a stable colloidal structure, with si ze and homogeneity

[0274] 20 inf luenced in a dose -dependent manner by the amount of encapsulated bioactive compound . In the absence of HTyr, the nanoparticles showed an average diameter of about 150 nm, a low polydispers ion index ( Pdl ) , indicative of good homogeneity, and a negative surface charge o f about -30 mV,

[0275] 25 a value considered optimal to ensure electrostatic stability of the system over time .

[0276] The addition of HTyr in the organic phase, in the range of 0 .2-2 mg / mL corresponding to 0 . 1-1 mg of bioactive compound per mg o f zein, resulted in a progressive increase

[0277] 30 in size and Pdl as a function of concentration, without however signi ficantly altering the zeta potential .

[0278] 22 Concentrations of 2 mg / mL or greater, however, caused marked colloidal destabilization, reflected in a significant increase in polydispersion, suggesting a critical encapsulation limit for hydrophilic compounds such as HTyr.

[0279] 5 The physical stability of the nanosystems was also assessed by the Turbiscan Lab®, with monitoring of the

[0280] Turbiscan Stability Index (TSI) at two temperatures (25°C and 37°C) . The formulations were stable at 25°C, except for the one containing 2 mg / mL HTyr, which showed a marked

[0281] 10 increase in TSI, indicative of instability.

[0282] At 37°C, a lowering of the slope of the TSI curves was observed, probably due to conformational rearrangements of the zein and more pronounced hydrophobic interactions, which may promote aggregation but also a new form of structural

[0283] 15 stabilization .

[0284] This result confirms the high property of nanomatrices to effectively retain the bioactive compound without originating sedimentation, flocculation or creaming phenomena over time.

[0285] 20 Figure 3 shows the results of the experimentation in terms of encapsulation efficiency and loading capacity.

[0286] The amount of HTyr contained in polymeric nanosystems was determined by Ultra -High-Performance Liquid

[0287] Chromatography (UHPLC) method, followed by High Resolution

[0288] 25 Mass Spectrometry (HRMS) with negative ionization, as previously described (Frisina et al., 2023) . Chromatographic separation was carried out using a Dionex Ultimate 3000 RS

[0289] (Thermo Scientific Rodano , MI, Italy) , equipped with a

[0290] Hypersil Gold C18 column (100 x 2.1 mm, 1.9 pm particle size,

[0291] 30 Thermo Scientific) , which was maintained at a temperature of

[0292] 23 24°C. The chromatographic column was balanced with 98% of solvent A (ultrapure water containing 0.1% formic acid) and

[0293] 2% solvent B (methanol) . The flow in column was maintained at 300 pl min i and the concentration of solvent B was

[0294] 5 increased linearly from 2% to 23% in 6 min, and maintained in isocratic mode for 5 min, then linearly increased from

[0295] 23% to 50% in 7 min, and from 50% to 98% in 5 min; subsequently, kept in isocratic mode for 6 min and finally brought back to 2% in 6 min and kept in isocratic mode for

[0296] 10 3 min . The UV / VIS detector has been set to 235, 254, 280,

[0297] 330 nm. The inj ection volume was 5 pl. The total chromatographic run time, including wash time and time for column balancing, was 38 minutes.

[0298] Mass determination was performed using a high-

[0299] 15 resolution Q-Exactive orbitrap mass spectrometer ( Thermo

[0300] Scientific, Rodano, MI, Italy) . Electrospray ionization

[0301] (ESI- Electrospray Soft Ionization) was selected in negative mode, with the following operating conditions : 70, 000 resolving power (defined as FWHM at m / z 200) , IT 100 ms, and

[0302] 20 ACG target 1*E6, Scan range (100-900 m / z) . In each scan, the exact negative mass [M-H] of the HTyr precursor

[0303] (153.0753 m / z) was selected in Parallel Reaction Monitoring

[0304] (PRM) mode. The MS / MS analysis was carried out according to the following operating conditions : resolution : 35, 000;

[0305] 25 Target AGC 1*E5; maximum IT 200 ms ; collision energy

[0306] ( stepped NCE) : 20,30,40. The isolation window of the quadrupole has been set to 2.0 m / z. The gas used as sheath gas (30 arb units) and auxiliary gas (10 arb units) was high- purity nitrogen. The instrument was calibrated before each

[0307] 30 analysis using a calibration solution provided by Thermo

[0308] Fisher Scientific.

[0309] 24 HTyr was characterized based on the corresponding HRMS spectrum, exact mass, characteristic fragmentations , and retention time. Data acquisitions and analysis, as well as instrumental controls were carried out using the Xcalibur

[0310] 5 software (version 4.1) . The HTyr bioactive compound was identified and quantified using an external calibration line in a range between 1.0 and 100 pg ml1, using a high-purity reference standard.

[0311] Formulations prepared with different amounts of HTyr

[0312] 10 (0.2-2 mg / mL corresponding to 0.1-1 mg of bioactive compound per mg of zein) were ultracentrifuged and the amount of HTyr encapsulated in the nanoparticles was determined as the difference between the amount of the bioactive compound added during the preparation of the nanosystems (Da) and the amount

[0313] 15 obtained from the analysis of the supernatant (De) , and expressed in %, according to the following equation:

[0314] A colloidal formulation without active ingredients was used as a "blank" during the analysis.

[0315] De

[0316] EE% = - x 100

[0317] Da

[0318] Loading capacity (LC%) was expressed as a percentage of

[0319] 20 the ratio of the total amount of encapsulated HTyr to the total weight of the nanoparticles according to the following equation :

[0320] 25

[0321] The amount of SD integrated into the colloidal particles was evaluated using a colorimetric assay.

[0322] 25 In Figure 3 it can be observed that the concentration of 1.6 mg / mL of bioactive compound was the maximum supported by zein-based colloidal systems, since exceeding this limit, the nano formulations showed a destabilization of the

[0323] 5 colloidal structure with consequent formation of sediments and macroaggregates, a phenomenon previously described in the DLS characterization phases.

[0324] Furthermore, as can be seen in Figure 3, nanospheres based on the natural polymer showed a direct proportionality

[0325] 10 between the increase in bioactive compound retention and its amount used in the preparation phases of formulations up to

[0326] 0.8 mg / mL; however, exceeding this value can be observed the saturation of the system. In detail, it is interesting to note that the system prepared with an initial concentration

[0327] 15 of HTyr equal to 1.6 mg / mL allowed to obtain an encapsulation efficiency of about 60%, corresponding to 0.96 mg / mL of bioactive compound actually retained in the system. This value is equivalent to about 0.48 mg of encapsulated HTyr per mg of zein (2 mg / mL) , showing a good retention capacity.

[0328] 20 In contrast, the use of lower initial concentrations of bioactive compound resulted in significantly reduced retention. Moreover, the evaluation of the loading capacity of the nanosystems revealed a remarkably high value (-25%) in the case of formulations prepared with 1.6 mg / ml of HTyr

[0329] 25 (Figure 3) , confirming the excellent ability of the natural biopolymer to effectively retain the bioactive compound.

[0330] RELEASE KINETICS

[0331] The applicant has also carried out release studies to support the efficacy of the formulation and matrix developed

[0332] 30 according to the invention.

[0333] 26 The kinetics of HTyr release from zein nanoparticles were evaluated by the dialysis method using Spectra / Por acetate cellulose membranes , with a cut-off of 10 kDa

[0334] (Spectrum Laboratories Inc. Netherlands) , sealed at the ends

[0335] 5 with two clips.

[0336] Htyr ril

[0337] Release (%) i x 100 Htyr load

[0338] In detail, 1 ml of particulate suspension was placed in the dialysis bag which was then transferred to a beaker containing 100 ml of release buffer (PBS, 0. IM pH 7.4) for a period of time equal to 160 hours (Gagliardi et al . ,

[0339] 10 2021a) . In parallel, the same test was conducted by placing the dialysis bag first in 100 mL of simulated gastric fluid

[0340] (SGF, 0.32% w / v pepsin, pH 1.2) and then transferred to 100 mL of simulated intestinal fluid (SIF, 1% w / v pancreatin, pH

[0341] 6.8) for 6 h. At predetermined time intervals, a s amp 1 e of

[0342] 15 liquid from the receptor solution (1 ml) was taken from the beaker and replaced with the same volume of fresh fluid. The samples obtained were analyzed using the HPLC method. The percentage of HTyr released was calculated with the previous equation wherein:

[0343] 20 HTyr ril amount of HTyr released at time t

[0344] HTyr load amount of HTyr encapsulated.

[0345] Release studies were conducted in triplicate.

[0346] The results obtained from the release tests showed a release of the encapsulated bioactive compound as a function

[0347] 25 of the simulated environment and the exposure time . In detail , in PBS it was possible to observe an almost negligible release profile of the bioactive compound, confirming the great affinity of the compound for the protein

[0348] 27 matrix ( Figure 5 ) . On the contrary, under conditions simulating digestion, i . e . using release buf fers enriched with pepsin and pancreatin, it is possible to observe a greater release of the bioactive compound; in detail , in

[0349] 5 figure 5 it is possible to appreciate a release of 7 % of encapsulated HTyr from the formulation prepared with 1 . 6 mg / ml of bioactive compound at pH 1 . 2 in the presence of pepsin, while at pH 6. 8 and in a buf fer containing pancreatin, the amount o f HTyr released by the same

[0350] 10 formulation was equal to 40% , revealing a more constant and protracted profi le over time . This i s due to the fact that zein tends to undergo faster digestion by pancreatin in intestinal fluid under alkaline conditions (Hurt ado -Lopez and Murdan, 2006 ) . In fact, the erosion of the polymer matrix

[0351] 15 at basic pH favours a greater release of the encapsulated active ingredient (Bhushani et al . , 2017 , Penalva et al . ,

[0352] 2015, Penalva et al . , 2015 ) . The same trend can be appreciated with the other formulations containing lower quantities of active ingredient , although it is interesting

[0353] 20 to note that the increase in the amount of HTyr contained in the protein matrix induces a lower release o f the bioactive compound over time ; in fact , the bioactive compound is released progressively and in inverse proportion to the concentration of the encapsulated compound .

[0354] 25 Taking into account the fact that the absorption of the bioactive compound takes place in the small intestine , the results described above provide the rationale for the use of zein-based nanosystems in order to implement the absorption of the compound after oral administration . In fact , the

[0355] 30 intrinsic mucoadhesive properties of the polymer, combined with the small s ize of the carriers , could be exploited to promote the absorption of particles ( and therefore of HTyr )

[0356] 28 through the intestinal epithelium, as already observed for other zein-based formulations ( Tran et al . , 2019 ) .

[0357] The results obtained show a controlled and pH- / enzyme- dependent release of HTyr from zein nanoparticles , with

[0358] 5 gradual release of the bioactive compound in simulated gastrointestinal conditions and negligible dif fusion in the gastric environment . Such experimental evidence is not present in the known technical documents . In particular, the system described in ( Panagiotopoulou et al . 2022 ) is designed

[0359] 10 for topical use, therefore free of requirements related to intestinal release or systemic bioavailability; moreover, it uses a synthetic DCA-HTyr hybrid or non-standardi zed crude extracts , without any evaluation of release under simulated physiological conditions . Telmoudi Arneni et al 2023 and

[0360] 15 Soleimanifar Matin et al 2019 , while employing matrices of natural origin (multicomponent and WPC-based, respectively) , do not propose targeted strategies for intestinal release , nor do they demonstrate the stability of the systems in the gastrointestinal environment . On the contrary, our system is

[0361] 20 characteri zed by : ( i ) experimentally confirmed selective and targeted release ; ( 11 ) smaller particles ( - 110 nm) , which represent an additional advantage for intestinal absorption, being documented that nanoparticles smaller than 150 nm can more ef fectively cross the intestinal epithel ial barrier ;

[0362] 25 ( ii i ) use of sodium deoxycholate as a stabili zer and promoter of absorption, thanks to its ability to mimic the action of intestinal bile salts . Also compared to the review Tapia-

[0363] Hernandez et al . , 2018 , which presents a general overview of zein-based systems , the present invention stands out for the

[0364] 30 use of an experimental formulation approach, validated, scalable , and designed to improve oral bioavailability .

[0365] 29 These characteristics constitute a clear technical advance and justify the value of the system described.

[0366] The applicant also performed analyses using Fourier transform infrared spectroscopy (FT-IR) .

[0367] 5 The vibrational spectra of zein, SD, HTyr, physical powder mixture, hollow zein-based or HTyr- containing nanosystems were detected using a Nicolet™ iS5 spectrometer coupled with an iD7 Attenuated Total Reflectance accessory

[0368] ( Thermo Fisher Scientific Inc . , Waltham, MA, USA) . The

[0369] 10 nanoparticles were freeze-dried without the use of cryoprotectants, before performing the analyses . FT-IR spectra were obtained through 64 acquisitions with a resolution of 4 cm" i in a wavelength range of 500 to 4000 cm"

[0370] The analyses were processed using the OMNIC software,

[0371] 15 version 9.12.1019. Each result reported is a representative analysis of three independent experiments.

[0372] For the evaluation of the antioxidant activity of the invention, the following experimental tests on cell cultures were carried out.

[0373] 20 Colon cancer cells (CaCo-2) were incubated in plastic culture discs (100 mmx20 mm) at 37°C (5% CO2) in a Forma®

[0374] Series II incubator (Water- Jacketed CO2 Incubator, Thermo

[0375] Scientific, Germany) using D-MEM enriched with Glutamax I, penicillin (100 pg / ml) , streptomycin (100 pg / ml ) ,

[0376] 25 amphotericin B (2.5 pg / ml) and fetal bovine serum (10% v / v

[0377] FBS) as a medium. The medium was changed every 48 hours. At the confluence of about 80%, the cells were treated with trypsin / EDTA (2 ml) , to separate them from the plate, and collected in a centrifuge tube containing 4 ml of culture

[0378] 30 medium necessary to buffer the trypsin present. The discs were then washed with 2 ml of PBS to remove the remaining

[0379] 30 cells, and everything was collected in the tube which was subsequently centrifuged at 1000 rpm for 10 minutes at room temperature (Eppendorf Centrifuge 5810) . The pellets obtained were resuspended in a suitable volume of medium and

[0380] 5 divided into discs or culture plates for subsequent in vitro tests . To evaluate the antioxidant activity of empty and

[0381] HTyr-containing nanosystems, the MTT test was performed in the presence of H2O2 2100 pM (Voci et al., 2022 ) . The cells were plated in 96-well multiwell (15xl03cells / 0.2 ml)

[0382] 10 treated with different concentrations of HTyr in free form or particle form (5, 10, 20 pM) for 24 h, and then subjected to oxidative stress by incubation with an aqueous solution of H2O2 (2100 pM, 1 h) (Gagliardi et al., 2021b) . 20 pL of tetrazolium salts , solubilized in a phosphate buffer

[0383] 15 solution (5 mg / mL) , were added to each well of the different plates, which were incubated again for 3 hours and analyzed with a spectrophotometer (xMARK™ Bio-Rad Laboratories Inc.,

[0384] Hercules , CA, USA) at a wavelength of 540 nm with reference to the wavelength of 690 nm. Untreated cells were used as a

[0385] 20 control .

[0386] Antioxidant activity was also evaluated by ORAC test.

[0387] The ORAC (oxygen radical absorbance capacity) test involves the use of a microplate fluorometer (Victor! multilabel counter, Perkin-Elmer Life Sciences, Waltham, MA;

[0388] 25 excitation 495 nm; emission, 535 nm, 0.5 sec for reading from the bottom of the plate, 1 reading per minute) , with

[0389] 24-well plates. A solution of PBS 10 mM (pH:7.4) was used as blank . 10 pM solutions of HTyr in free form or encapsulated in zein nanoparticles and empty nanosystems at

[0390] 30 the same concentration as those containing the bioactive compound were analyzed. In each well 80 pl of sample or blank

[0391] 31 or Trolox (6.25, 12.5, 25 and 50 pM in PBS) was added to 800 pl of a disodium fluorescein solution (FL, 14pM in PBS, preincubated at 37 °C for 15 min) . Each solution was analyzed in duplicate in a " forward- then-reverse" order as already

[0392] 5 described in the literature (Ou, Hamps ch-Woodi 11 & Prior,

[0393] 2001) . The reactions were triggered by the addition of 300 pL of AAPH (31.7 mM in PBS) . The temperature was set to 37°C.

[0394] The final values of ORACFL were calculated using a regression line obtained by interpolation of Trolox concentrations with

[0395] 10 the corresponding net areas under the FL decay curve (Prior et al . , 2003) :

[0396] Y(pM Trolox) = a + bX(AUC_n) and were expressed as Trolox equivalents (pM) .

[0397] The net area under the curve was calculated as:

[0398] 15 wherein fo represents the initial fluorescence reading, fi is the reading at time i.

[0399] The net AUG was obtained as: AUCsampie-AUCbiank .

[0400] According to one aspect of the invention, the formulation based on HTyr and its derivatives, encapsulated

[0401] 20 in zein-based nanosystems , prepared with SD, contains variable concentrations of HTyr.

[0402] PARTICLES CHARACTERIZATION

[0403] According to one aspect of the invention, the formulation comprises zein nanospheres with an average

[0404] 25 diameter preferably equal to about 150 nm, a low polydispersion index (Pdl) value and a surface charge of

[0405] 32 about -30 mV, an ideal value for the stability of systems over time .

[0406] According to one aspect of the invention, the addition of HTyr in the organic phase , in the concentration range

[0407] 5 0 .2 -2 mg / mL, advantageous ly induces an increase in si ze and

[0408] Pdl proportional to the concentration of the active ingredient used, while not influencing the surface load .

[0409] It was experimentally found that bioactive compound concentrations equal to or greater than 2 mg / mL favored a

[0410] 10 signi ficant destabili zation of the colloidal structure with a consequent increase in polydispersion, suggesting the inability of the systems to ef fectively retain the hydrophilic compound .

[0411] Figure 1 shows the average si ze , polydispersion index

[0412] 15 and zeta potential of zein nanoparticles prepared with 2 mg / mL polymer , 1 .25% w / v sodium deoxycholate and di f ferent concentrations of HTyr .

[0413] During the chemi cal -physical characterization of a colloidal drug deli very system, temperature -dependent

[0414] 20 stability is a very important parameter to evaluate . The analysis , in the experimentation conducted by the Appl icant on the formulation according to the invention, was carried out by means of Turbiscan Lab® Expert , which correlates the transmittance and backsca t tering of a sample as a function

[0415] 25 of time and temperature and the data obtained were expressed as a Stabi lity Index ( TS I ) . The advantage of this instrument , compared to conventional methods of analysis , lies in the abi lity to quickly determine the stability of a colloidal system with a high degree of accuracy, without any mechanical

[0416] 30 destruction or manipulation of the sample .

[0417] 33 The nanospheres were incubated at room temperature

[0418] (20°C) and body temperature (37°C) to assess the influence of the hydrophilic compound on their stability.

[0419] As can be seen in Figure 2, zein nanospheres showed no

[0420] 5 significant changes in their profiles after encapsulation of the active substance at 25°C, with the exception of the formulation containing 2 mg / ml HTyr which showed a significant increase in TSI values, suggesting that a higher amount of active ingredient may promote structural

[0421] 10 destabilization .

[0422] On the contrary, the increase in temperature (37°C) promoted a slight decrease in the slopes of the TSI curves of the various formulations ( Figure 2 ) . This result is probably due to a rearrangement of the protein that exposes

[0423] 15 the reactive sites to temperatures higher than ambient temperatures, promoting the aggregation of molecules through hydrophobic interactions.

[0424] This result confirms the high property of nanomatrices to effectively retain the bioactive compound without

[0425] 20 originating sedimentation, flocculation or creaming phenomena over time.

[0426] Figure 2 shows the Turbiscan Stability Index (TSI) of zein formulations (2 mg / mL polymer, 1.25% w / v SD) prepared with 0.2-2 mg / mL HTyr. The analyses were carried out at 25°C

[0427] 25 (A) and 37°C (B) for one hour.

[0428] Another parameter to be evaluated in the nanoparticle characterization phases concerned the encapsulation efficiency of the active ingredient, a necessary factor for subsequent in vitro trials, determined by UHPLC-HRMSS.

[0429] 34 In detail , different concentrations of the compound were used in order to evaluate the retention capacity of the polymer matrix; in Figure 3 it can be observed that the concentration of 1.6 mg / mL of bioactive compound was the

[0430] 5 maximum supported by zein-based colloidal systems , since exceeding this limit, the nano formulations showed a destabilization of the colloidal structure with consequent formation of sediments and macroaggregates, a phenomenon previously described in the DLS characterization phases.

[0431] 10 Furthermore, as can be seen in Figure 3, nanospheres based on the natural polymer showed a direct proportionality between the increase in bioactive compound retention and its amount used in the preparation phases of formulations up to

[0432] 0.8 mg / mL; however, exceeding this value can be observed the

[0433] 15 saturation of the system.

[0434] In detail, it is interesting to note that the system prepared with an initial concentration of HTyr equal to 1.6 mg / mL allowed to obtain an encapsulation efficiency of about

[0435] 60%, corresponding to 0.96 mg / mL of bioactive compound

[0436] 20 actually retained in the system. This value is equivalent to about 0.48 mg of encapsulated HTyr per mg of zein (2 mg / mL) , showing a good retention capacity of the particle system. In contrast, the use of lower initial concentrations of bioactive compound resulted in significantly reduced

[0437] 25 retention . In particular, nanoparticles prepared with 0.2 mg / ml , 0.4 mg / ml, 0.8 mg / ml and 1.2 mg / ml showed an encapsulation efficiency of 20% (0.02 mg of encapsulated

[0438] HTyr / mg of zein) , 47% (0.095 mg of encapsulated HTyr / mg of zein) , 59% (0.23 mg of encapsulated HTyr / mg of zein) and 58%

[0439] 30 (0.34 mg of encapsulated HTyr / mg of zein) , respectively.

[0440] 35 Moreover, the evaluation of the loading capacity of the nanosystems revealed a remarkably high value (~25%) in the case of formulations prepared with 1.6 mg / ml of HTyr (Figure

[0441] 3) , confirming the excellent ability of the natural

[0442] 5 biopolymer to effectively retain the bioactive compound.

[0443] Figure 3 then shows the evaluation of the encapsulation efficiency (EE%) and loading capacity (LC%) of HTyr in zein polymer matrices as a function of the amount of active ingredient used in the preparation phases of the nanosystems.

[0444] 10 Infrared (IR) spectroscopy is the technique chosen to evaluate the chemical interactions between the molecules that make up the formulation of zein and HTyr. The FT-IR spectra of zein, SD, HTyr, their physical mixture, hollow and containing the active ingredient nanoparticles are

[0445] 15 represented in Figure 4. The FT-IR spectrum of HTyr showed the following characteristic peaks: 3216 cim1due to O-H stretching vibrations, the 2938 cm i band associated with the vibrational mode of aromatic C-H bond stretching. The bands associated with the double bond between the C=C aromatics

[0446] 20 are shown at 1607 cm" i and 1441 cm1However, the most significant bands in the HTyr spectrum (as is the case with all aromatic compounds) are visible at low frequencies. These bands associated with intense absorption result from the out-of-plane bending of the C-H bonds of the substituted

[0447] 25 benzene compound (at positions 1, 2 and 4) at 868, 810, 714 and 686 cm i

[0448] Zein showed the typical peaks of amide I and IT at 1640 and 1515 cm" i respectively. The first peak was attributed to

[0449] C=O stretching of the carboxyl group while the one at 1515

[0450] 30 cm" i was attributed to N-H bending and C-N stretching vibrations . In addition to showing a peak at 3252 cm i due

[0451] 36 to hydroxyl stretching, the SD showed another absorption band at 1580 cm" i due to COO groups ( Gagliardi et al . , 2021 ) .

[0452] As can be seen in Figure 4 in empty nanoparticles the absorption band of the zein ' s O-H vibration at 3282 cm" i

[0453] 5 shi fts to 3294 cm in addition, the peaks of amides I and

[0454] I I of the protein undergo a further shi ft from 1640 to 1654 and from 1515 to 1550 cm" i respectively . These results showed that the predominant interactions in nanosystems are electrostatic in nature and can be traced back to the

[0455] 10 hydrogen bonds that are established between the protein and

[0456] SD . Interestingly, in the spectrum of HTyr- containing nanoparticles , the absorption bands of hydroxy tyrosol decrease or even disappear ; this is a clear indication of the overlap of the characteristic peaks of HTyr with the

[0457] 15 absorption bands of the polymer matrix, contrary to what was found by the analysis of the physical mixture of powders wherein the peculiar peaks of the bioactive compound are distinguishable, albeit in a less marked way .

[0458] Subsequently, release studies were conducted in order

[0459] 20 to evaluate the release of HTyr over time ( Figure 5 ) ; in particular , the analysis was carried out by selecting dif ferent buf fers . In detail , in PBS it was possible to observe an almost negligible release profi le of the bioactive compound, confirming the great af finity of the compound for

[0460] 25 the protein matrix ( Figure 5 ) . Similar results have already been observed for other zein nanosystems containing dif ferent bioactive compounds ( Gagliardi et al . , 2021 ) . On the contrary, under conditions simulating digestion, i . e . using release buffers enriched with pepsin and pancreatin,

[0461] 30 it is poss ible to observe a greater release of the bioactive compound; in detail , in figure 5 it is possible to appreciate a release of 7% of encapsulated HTyr from the formulation

[0462] 37 prepared with 1.6 mg / ml of bioactive compound (corresponding to 0.48 mg of encapsulated HTyr / mg zein) at pH 1.2 in the presence of pepsin after 2 h, while at pH 6.8 and in a buffer containing pancreatin, the amount of HTyr transferred by

[0463] 5 the same formulation was 40% after 6 h, revealing a more constant and protracted profile over time. This is due to the fact that zein tends to undergo faster digestion by pancreatin in the intestinal fluid under alkaline conditions

[0464] (Hurtado-Lopez and Murdan, 2006) . In fact, the erosion of

[0465] 10 the polymer matrix at basic pH favours a greater release of the encapsulated active ingredient (Bhushani et al., 2017,

[0466] Penalva et al., 2015, Penalva et al., 2015) . The same trend can be appreciated with other formulations containing lower quantities of active ingredient, although it is interesting

[0467] 15 to note that the increase in the amount of HTyr contained in the protein matrix induces a lower release of the bioactive compound over time ; in fact, the bioactive compound is released progressively and in inverse proportion to the concentration of the encapsulated compound. In particular,

[0468] 20 and as shown by the graphs in figure 5, in the gastric environment all zein nanoparticles , regardless of the initial concentration of HTyr, showed an almost similar percentage of release of the encapsulated bioactive compound, between 7% and 12%, after 2 hours of incubation.

[0469] 25 On the contrary, in the intestinal environment, the increase in the concentration of HTyr led to a more gradual and controlled release . After 6 hours, nanoparticles prepared with 0.2, 0.4, 0.8, and 1.2 mg / mL HTyr (equal to 0.02, 0.095,

[0470] 0.23, and 0.34 mg of encapsulated HTyr per mg of zein) showed

[0471] 30 an intestine release rate of 70%, 60%, 51%, and 48%, respectively. These results confirm an inverse relationship

[0472] 38 between the degree of encapsulation and the percentage of intestinal release of the bioactive compound .

[0473] Taking into account the fact that the absorption of the bioactive compound takes place in the small intestine , the

[0474] 5 results described above provide the rationale for the use of zein-based nanosystems in order to implement the absorption of the compound after oral administration . In fact , the intrinsic mucoadhesive properties of the polymer, combined with the small s ize of the carriers , could be exploited to

[0475] 10 promote the absorption of particles ( and there fore of HTyr ) through the intestinal epithelium, as already observed for other zein-based formulations .

[0476] The antioxidant properties of the formulations described on the CaCo-2 cel l l ine , used as a re ference model

[0477] 15 to mimic the intestinal epithelium in vi tro, were investigated as a function of the amount of HTyr used .

[0478] As shown in Figure 6 , the nanoencapsulation of HTyr in zein-based systems allowed to obtain a greater protective effect than the free bioactive compound at all the

[0479] 20 concentrations investigated .

[0480] Furthermore , in the experimentation conducted by the

[0481] Applicant it emerged that the protein nanocarrier showed an antioxidant ef fect even in the absence of HTyr ; this is probably due to the peculiar chemi cal -physical

[0482] 25 characteri stics of the polymer .

[0483] In fact, several studies in the l iterature have revealed that some amino acid res idues or short peptide chains present in the protein are directly correlated with the scavenger activity of free radicals ( Chan et al . , 1994 , Hernandez-

[0484] 30 Ledesma et al . , 2007 ) .

[0485] 39 In detail, it has been attributed to the presence of amino acid residues such as His, Arg, Ala, Vai, Met and Leu which have been shown to possess a predominant antioxidant activity both in free form and as residues in peptides and

[0486] 5 proteins .

[0487] For this reason, the greater efficacy of zein nanoparticles containing HTyr is advantageously due to a synergy of action between the intrinsic antioxidant activity of the nanoparticle and the pharmacological action of the

[0488] 10 active ingredient.

[0489] These data were further confirmed by the ORAC test. The method consists of providing a controlled source of peroxyl radicals that simulates their reactions with lipids, thus allowing to estimate the antioxidant action in foods and

[0490] 15 physiological systems. The antioxidant capacity was measured using fluorescein (FL) as a fluorescent probe: the inhibition of peroxyl radical-induced oxidation of FL, activated by the thermal decomposition of 2,2' -azobis (2- methylpropionamidine) dihydrochloride (AAPH) , was evaluated

[0491] 20 as already validated in the literature (Ou et al., 2001;

[0492] Prior et al., 2003) .

[0493] As can be seen from Table 1, the empty formulation did not show high antioxidant activity, probably due to the reduced amount of protein used for the assay. On the contrary, the formulation containing HTyr showed a pronounced antioxidant activity, comparable to that obtained by the use of the bioactive compound in free form (Ou et al., 2001, Nardi et al . , 2014) .

[0494] 40 The results obtained further confirm the ability of the protein matrix to preserve the antioxidant activity of the bioactive compound .

[0495] Table 1 : GRACED values of HTyr, zein nanoparticles and HTyr-containing nanosystems .

[0496] Sample ( 10 pM) ORACFL3

[0497] HTyr 61 . 69

[0498] Zein Nps 1 . 10

[0499] HTyr-Zein Nps 69 . 62 a Data expressed as the ±StD mean of three independent experiments (pmol Trolox I-1)

[0500] Advantageously according to the invention, zein is

[0501] 5 eas ily used to develop colloidal formulations , through the use of green solvents and simple methods . The versatility of the protein, associated with its mucoadhesive properties , easy availability and low cost , are j ust some of the properties that make this material an excellent candidate

[0502] 10 for the reali zation of innovative nanoparticle systems .

[0503] Advantageously according to the invention, the encapsulation of compounds of natural origin in the protein matrix al lows to obtain new nutraceutical formulations and / or forti fied food products .

[0504] 15 Advantageously, the nanoparticles according to the invention are able to gradually release hydroxy tyro sol , tyrosol and their derivatives in simulated gastric and simulated intestinal environments .

[0505] Advantageously, the nanoparticles according to the

[0506] 20 invention are able to preserve the antioxidant action of hydroxytyrosol , tyrosol and their derivatives .

[0507] 41 Advantageously, the formulation according to the invention is ef fective and safe for nutraceutical and / or pharmaceutical use , in particular as an antioxidant .

[0508] Finally, it is clear that modi fications and variants

[0509] 5 can be made to the formulation based on hydroxy t yroso 1 , tyrosol and their derivatives nanoencapsulated in a protein matrix according to the invention without leaving out of the protective scope of the present invention, as defined in the attached claims .

[0510] 10 The invention described has multiple technical , technological and application advantages that make it innovative and superior to conventional formulations for the release of bioactive compounds .

[0511] First of al l , the choice to use zein, a vegetable

[0512] 15 protein with GRAS status , makes it poss ible to obtain biocompatible , biodegradable nanoparticles suitable for oral use . The system is free of synthetic polymers , toxic solvents or multi-component components that are di f ficult to standardise, making it perfectly compatible with the

[0513] 20 nutraceutical and pharmaceutical industries in terms o f safety, sustainability and reproducibility . In addition, zein is known for its excellent properties to protect the active ingredient against light -induced degradation, pH changes and critical environmental conditions , contributing

[0514] 25 to the stability of the encapsulated compound during storage and passage through the gastrointestinal tract .

[0515] The preparation technique , based on nanoprecipitation, is s imp 1 e , scalable, easily trans ferable in GMP production environments and allows to obtain highly monodispersed

[0516] 30 nanoparticles ( PDI < 0 . 2 ) , small in si ze ( about 100-150 nm) , and stable over time . These characteristics are essential to

[0517] 42 ensure absorption in the intestine: colloidal systems with a size of less than 200 nm have been shown to be able to cross epithelial barriers more efficiently than larger particles, improving the systemic bioavailability of

[0518] 5 encapsulated active ingredients (Desai et al., 1996; He et al., 2012; Banerjee et al., 2016) .

[0519] In addition to this is the inclusion of sodium deoxycholate (SD) as an anionic and functional surfactant, which plays a dual role: on one hand, it stabilizes colloidal

[0520] 10 dispersion by preventing the formation of aggregates, and on the other hand, it acts as a promoter of intestinal absorption. As an endogenous bile salt, sodium deoxycholate promotes trans cellular and paracellular permeation and facilitates the reversible opening of tight junctions

[0521] 15 (Mo ghimipour et al . , 2015; Mikov et al . , 2006) . This contributes to efficient and selective absorption of the active ingredient encapsulated in zein nanoparticles, improving their systemic availability.

[0522] In addition, the proposed system demonstrated

[0523] 20 considerable formulation versatility, having been successfully tested on both hydrophilic and lipophilic compounds . The ability of the zein matrix to effectively encapsulate hydroxytyrosol and its natural derivatives has been confirmed experimentally, with an encapsulation

[0524] 25 efficiency >60% and a loading capacity of up to 25%, results rarely achievable with simple protein matrices.

[0525] The stability of the formulations has been experimentally validated by advanced techniques (DLS,

[0526] Turbiscan) , even under stress conditions (temperature, pH,

[0527] 30 presence of enzymes) . The system also shows a controlled, selective and pH- / enzyme-dependent release, with gradual

[0528] 43 release in a simulated intestinal environment and minimal dif fusion in a gastric environment . This profile is particularly relevant for the optimi zation of the systemic bioavailability of polyphenolic compounds , which are known

[0529] 5 to be unstable and rapidly metaboli zed if not appropriately conveyed . This is particularly relevant in pathological conditions wherein oxidative stress plays a central role . In the cardiovascular f ield, targeted release of hydroxytyrosol can help reduce LDL oxidation, slow down the progression of

[0530] 10 atherosclerosis , and improve endothelial function . In neurodegenerative diseases , the protection of neurons from damage induced by reactive oxygen species (ROS ) can delay the processes of neuroinflammation and neurodegeneration, promoting cell survival . In the metabolic field, in

[0531] 15 particular in diabetes mellitus and its microvascular complications ( retinopathy, nephropathy, neuropathy) , the antioxidant activity of the system can reduce glycotoxicity and improve the redox balance in damaged tissues .

[0532] In chronic inflammatory bowel diseases , the protection

[0533] 20 of the active ingredient in the gastric environment and the targeted intestinal release make it poss ible to locally counteract inflammation and the accumulation of ROS in the intestinal mucosa, with beneficial ef fects in the control of tis sue damage . In addition, the system shows potential

[0534] 25 application in autoimmune diseases ( e . g . rheumatoid arthritis and systemic lupus erythematosus ) , wherein the regulation of oxidative stress can reduce immune activation and the progres sion of systemic damage . In oncology, hydroxytyrosol is known for its ability to interfere with

[0535] 30 cel l proli feration and modulate redox homeostasis in tumor tis sues ; the use of zein nanoparticles would allow for more eff icient and targeted delivery, suggesting possible

[0536] 44 applications in chemoprevention or in support of conventional anticancer approaches .

[0537] Finally, also in the dermatological and cosmetic fields , the formulation could of fer protection against UV-

[0538] 5 induced free radical damage, with potential benefits in premature skin aging .

[0539] Overall , the developed system represents an advanced, functional and industrially promising technological solution for the oral delivery of thermolabile and pH-sensi five

[0540] 10 bioactive compounds . I t fully meets the requirements of patentabil ity in terms of novelty, inventive step and industrial applicability .

[0541] 45

Claims

CLAIMS1 . Protein nanoparticles comprising a bioactive compound chosen from hydroxytyrosol , tyrosol , olive phenolic compounds , and mixtures thereof , and a zein protein5 matrix stabili zed with sodium deoxycholate .2 . The protein nanoparticles according to the previous claim, having an average diameter between 100 nm and200 nm, preferably between 140 nm and 160 nm and even more preferably equal to about 150 nm, the diameter10 measurement being performed with photon correlation spectroscopy, us ing a Zetasi zer Nano ZS spectrophotometer from Malvern Instruments , UK .

3. The nanoparticles according to anyone of claims 1-2 capable of releasing <12% , preferably between 7- 12 % , of15 the bioactive compound content in a gastric environment simulated at pH 1 . 2 in a time frame of about 2 hours , and >48 % , preferably between 48 -70% of said content in a simulated intestinal environment at pH 6 . 8 , in a time frame of about 6 hours .20 4 . A formulation comprising protein the nanoparticles according to anyone of claims 1-3 and a pharmaceutically or nutraceutically acceptable agent .5 . The formulation according to the previous claim in freeze-dried form to be reconstituted or in liquid25 dispersion form, preferably aqueous dispersion .

6. The formulation according to anyone of claims 4-5 which is a nutraceutical composition .7 . The formulation according to anyone of claims 4-5 which is a pharmaceutical composition .30 8. The nanoparticles according to anyone of claims 1-3 and the formulation according to claim 7 for use in diseases caused by oxidative damage and benefiting from the46administration of effective amounts of said nanoparticles and formulations.

9. The nanoparticles and formulation according to the previous claim for use in the following conditions:5 cardiovascular disorders, atherosclerosis, hypertension, myocardial ischemia; neurodegenerative diseases, Alzheimer's, Parkinson's, amyotrophic lateral sclerosis; diabetes mellitus and related complications, neuropathy, nephropathy, retinopathy; chronic10 inflammatory bowel diseases, Crohn ' s disease, ulcerative colitis ; autoimmune diseases , rheumatoid arthritis , systemic lupus erythematosus ; metabolic diseases, metabolic syndrome, non-alcoholic fatty liver disease; skin aging and free radical damage induced by15 UV radiation; solid tumors and neoplasms associated with chronic oxidative stress.

10. A process for the preparation of nanoparticles according to anyone of claims 1-3 comprising the following basic steps:20 1) Preparing an organic phase at room temperature wherein an aliquot of zein is added to a hydroalcoholic solution to have a concentration of 1.5-2.5 mg / ml , preferably an ethanol / water solution (2:1 v / v;25 ID Preparing an aqueous solution of sodium deoxycholate monohydrate at room temperature, preferably at a concentration of 1-2% w / v; ill) Adding an aliquot of a bioactive compound ingredient chosen from hydroxytyrosol, tyrosol,30 phenolic compounds from the olive tree , and mixtures thereof to the organic phase prepared in step i) to have a mixture i ) -A or to the47hydrophilic solution of step ID to have a mixture ii)-B depending on the lipophilic or hydrophilic nature of the bioactive compound; iv) Mixing the organic phase of step i) with mixture5 ii) -B or mixture i)-A with the sodium deoxycholate solution of step iii) in a ratio of1-3:5-10 (v / v) , respectively; v) Evaporating the organic phase, preferably alcohol, present in the mixture of step iv) at10 a temperature below 37 °C and under stirring to form the nanoparticles; vi) Purifying the nanoparticles thus obtained.11 . A process according to the previous claim wherein the nanoparticles obtained are subjected to freeze-15 drying in the form of a powder to be reconstituted.48

Images