**3. Antioxidants**

Reactive oxygen species (ROS) is a term used for oxygen containing free radicals (such as O2 •, HO•, HO2 •, RO•, ROO•) or reactive oxygen-containing compounds (such as H2O2, O3, 1O2), depending on their reactivity and oxidizing ability. ROS participate in diverse chemical reactions (oxidative stress) resulting in the decomposition of biologically active compounds or biomolecules. Antioxidants that are able to protect other molecules from the damaging effects of such ROS can be used as excipients in formulations or as biologically active compounds preventing oxidative stress [131]. Bioactive compounds like polyphenols, flavonoids, and vitamins showing antioxidant properties are suitable to be used for the fortification of food products to enhance their functionality, and therefore, encapsulation systems for the delivery of such nutraceuticals are necessary to overcome their low stability and bioavailability [132]. The choice of the appropriate encapsulation method is essential, because the modification of bioactivity (increase, preservation, or decrease) is affected by interactions established between the functional groups of the encapsulated compound and the encapsulating nanomaterial [133].

#### *3.1. Nanoformulations with Antioxidant Capacity*

A significant property of nanoformulations is the possibility to co-encapsulate antioxidants together with an active ingredient and thus increase the stability and extend expiration. As antioxidant excipients, PUFAs, carotenoids, antioxidant plant extracts, CUR, and catechins can be used, and as formulations, nano/micro emulsions, NLCs, NPs/MPs, and liposomes can be applied.

The antioxidant capacity of α-LNA loaded microemulsion was strongly enhanced after the introduction of carbon dots, which were distributed mainly at the oil-water interface, suggesting a "turn off" effect of the interface [134]. Benzylisothiocyanate nutraceutical encapsulated in a stable α-tocopherol-based O/W NE stabilized with a nontoxic, biodegradable surfactant, sodium stearoyl lactate, showed better antioxidant activity than pure and CUR encapsulated NEs, however CUR entrapped in the NE was effectively protected from UV light-induced degradation [135].

AST-loaded NLCs with the Z-average size of 94 nm containing α-tocopherol and EDTA as antioxidants that were stabilized using Tween 80 and lecithin and mixed with non-pasteurized CO2-free beer at the volume ratio of 3:97 showed improved stability at low storage temperature of 6 ◦C [136].

Anionic sphere-shaped core-shell NPs with zein-epigallocatechin gallate (EGCG) conjugates as the hydrophobic core and a biosurfactant (rhamnolipid) as a shell with average diameters <200 nm co-loaded with CUR and RES protected these nutraceuticals from degradation, simultaneously preserving their antioxidant activity, and by mixing these NPs with lipid droplets, the bioaccessibility of both encapsulated compounds pronouncedly increased [137]. The incorporation of polysaccharides as a second polymer matrix can provide stability in zein NPs used as DESs for antioxidants in the prevention of chronic degenerative diseases [138].

Ethyl cellulose MPs with encapsulated hydroxytyrosol, a constituent of olive oil showing antioxidant properties, produced by double emulsion solvent evaporation (average particle size ranging from 156.6 ± 6.9 μm to 304.0 ± 16.0 μm) demonstrated the effectiveness of their gastro-resistance and the antioxidant capacity preservation of >50%, indicating possible applications of this formulation in foods, drugs, and nutraceuticals [139]. *Citrus reticulata* Blanco cv. unshiu peel extract (CPE) flavonoids encapsulated by pectin NPs with particle size 271.5 ± 5.3 nm released only 28.78% of flavonoids in simulated gastric fluid within 2 h compared to naked CPE and showed higher antioxidant activity than blank pectin NPs and free CPE [140]. The replacement of 30% of pectin (low charge density) with alginate (high charge density) forming shell around zein NPs significantly improved the aggregation stability at pH 5–7 and high ionic strengths (2.0 μM NaCl), and CUR encapsulated in these core-shell NPs was characterized by higher antioxidant and radical scavenging activities than CUR dissolved in ethanol solutions [141].

The comparison of liposomes and CS coated liposomes co-loaded with vitamin C and FA with the mean particle size of 138 nm and 249 nm, respectively, showed the higher EEs of both drugs as well as the higher antioxidant activity of CS coated liposome nanoformulation, suggesting that it could be applied as a promising DES in the food industry [142]. The deposition of CS and alginate layers on CUR NEs improved CUR antioxidant capacity during in vitro digestion and showed a better control of the rate and extent of lipid digestibility by decreasing free fatty acids release compared to uncoated NEs [143].

#### *3.2. Supplements with Antioxidant Effect*

Oxidative stress is able to generate an imbalance between the production and accumulation of ROS in cells and tissues and thus modify the ability of a biological system to detoxify these reactive products. ROS have several physiological roles (i.e., cell signaling), and they are normally generated as by-products of oxygen metabolism; despite this, environmental stressors (i.e., UV, ionizing radiations, pollutants, and heavy metals) and xenobiotics (i.e., antiblastic drugs) contribute significantly to ROS production, thus causing an imbalance that leads to cell and tissue damage (oxidative stress) [144,145]. At present, various oral (flavonoids, carotenoids, vitamin C, and synthetic) antioxidants, see Figure 2, are available on the market and are generally recommended to be used. However, these supplements should be used in accordance with recommendations of a conscious physician or health care professional to avoid their adverse effect—pro-oxidant activity depending on the specific set of conditions (their dosage, redox conditions, the presence of free transition metals in cellular milieu) [144–148]

**Figure 2.** Most frequently used human dietary nanosupplements.

An improvement in aqueous solubility, antioxidant and other health-promoting properties, in vitro GI release profile, and protection against process and environment harsh conditions (e.g., light, oxygen, high temperatures, and humidity) of hydrophobic food bioactive compounds suitable as DISs could be achieved by nanoencapsulation using different nanoencapsulation DESs, including inclusion complexes of CDs, amylose, yeas<sup>t</sup> cells, nanogels, NEs, nanofibers, nanosponges, nanoliposomes, and NPs made with lipids [149].

A CUR-β-CD inclusion complex and iron oxide NPs were co-encapsulated in liposomes, and these CUR-in-β-CD-in-nanomagnetoliposomes with mean particle size 67 nm and 71% CUR EE showed a radical scavenging property exceeding that of conventional CUR liposome and iron oxide NPs [150].

β-Car, the most important dietary source of provitamin A, is necessary for optimum human health. To increase its solubility and bioaccessibility, 0.1% β-Car was dispersed in corn oil (5 or 10%) and homogenized with 2% SCas solution at 100 MPa, and the prepared NEs had particle sizes <200 nm. β-Car stability towards oxidation decreased with the decreasing droplet diameter, and the extent of lipolysis in an in vitro system was higher and linearly related to the inverse of the droplet diameter [151]. The updated understanding of emulsion-based DESs for β-Car was overviewed by Mao et al. [152] who focused their attention also on emulsion design enabling the delivery of β-Car in complex food systems and fulfilling its benefits in functional foods. Lipid droplets in β-Car enriched O/W emulsions stabilized with surface-active chlorogenic acid-lactoferrin-polydextrose conjugate used as an emulsifier with the mean particle diameter of <400 nm across the pH range 2–9 (except pH value around 6.0) exhibited better stability against droplet aggregation under simulated GI tract conditions (mouth, stomach, and small intestine) than other systems, which resulted in improved β-Car bioaccessibility, and such formulations could be potentially applied as protectors and carriers of hydrophobic drugs, supplements, and nutraceuticals [79]. Encapsulation of β-Car into solid lipid MPs of palm stearin stabilized with hydrolyzed soy protein isolate and containing α-tocopherol with the mean diameter of about 1.2 μm preserved approx. 75% of the encapsulated β-Car after 45 d of storage, and the formulation withstand treatments with higher temperatures (>60 ◦C), while showing low stability after different ionic strength stresses [153].

DESs of Q, one of the most well-known flavonoids that was included in human diet long ago due to health benefits associated with its antioxidant, anti-inflammatory, antiviral, and anticancer activities as well as Q biological activities themselves, its chemical stability, metabolism, and positive impact on some cardiovascular diseases (CVD) (i.e., heart disease, hypertension, and high blood cholesterol) were overviewed by Wang et al. [154]. An extract of tartary buckwheat rich in flavonoids (TBFs) incorporated in spherical biocompatible lipid–polymer hybrid NPs of 61.25 ± 1.83 nm showed higher antioxidant activity and significant suppression of the pro-inflammatory cytokine secretion in RAW 264.7 macrophage compared to free TBFs and exhibited immune-enhancing efficacy in immunosuppressed mice, suggesting that such nanosystem loaded with TBFs is suitable for nutraceutical applications [155]. Nanoliposomes incorporating olive leaf extract containing high levels of phenolic compounds and oleuropein, showing antioxidant and antimicrobial activities, with average particle size 25–158 nm, negative charge, and EE 70.7–88.2%, which were supplemented to yogurt, improved its antioxidant activity, and no significant changes in color and sensorial attributes were observed, suggesting that olive leaf phenolics can be entrapped in nanoliposomes and could increase the nutritional value of products like yogur<sup>t</sup> [156].

RES encapsulated in zein/pectin core-shell NPs with mean diameter approx. 235 nm and polydispersity index 0.24 showed improved in vitro antioxidant activity as well as lower IC50 values (by 32%) related to antiproliferative activity tested using human hepatocarcinoma Bel-7402 cells compared to free RES, suggesting that such nanoformulation of RES could be used in functional foods and beverages as well as in DISs and pharmaceutical products [157]. The aqueous solubility of RES from α-lactalbumin ( α-Lalb)-RES nanocomplexes was 32-fold higher than that of free RES, and the nanocomplexes considerably improved the antioxidant chemical stability under storage, especially at pH 8.0 and high temperature, and showed superb in vitro antioxidant activity compared to free RES, suggesting that α-Lalb as a nanoscale carrier could effectively deliver lipophilic nutraceuticals in the functional food, biomedical, and pharmaceutical products [158]. The protection of light-sensitive AST, a carotenoid with the most potent antioxidant activity, from photodegradation achieved by its inclusion in different hierarchically assembled nano- and microstructures in order to produce model foods for humans and fishes decreased as follows: NEs > carrageenan-coated NEs > CS coated NEs, and CS beads provided higher protection to AST than alginate beads. These hierarchically assembled materials represent ideal platforms to create foods for humans and animal species, because their flexibility enables also the incorporation of other active molecules such as proteins, PUFAs, antibiotics, antiparasitics, etc. [159]. The average size (94 nm) of AST-loaded NLCs containing α-tocopherol and ethylenediaminetetraacetic acid as antioxidants and stabilized with Tween 80 and lecithin increased at pH ≤ 5, high NaCl concentrations ( ≥50 mM), and slightly at simulated gastric juice, which was connected mainly with decreasing zeta-potential, while increasing at treatment at 80/90 ◦C. On the other hand, cryoprotectant glycerol prevented the aggregation of AST-NLCs during freeze-thawing. Therefore, this nanoformulation could be used as a DIS [160].

Nanoscale thymoquinone [161], which was found to improve the anticancer roles of doxorubicin by upregulation of P53 and downregulation of Bcl2 and potentiate paclitaxel's apoptosis in MCF-7 breast cancer cells, could protect also against diabetes, inflammation, central nervous system, and hepatotoxicity primarily by enhancing the antioxidant status of organs and could be considered as a promising nutraceutical for human health [162].

It should be noted that microencapsulation of riboflavin, a water-soluble vitamin acting as cofactor in various processes of oxidation-reduction in a cellular system, with galactomannan biopolymer and Pluronic® F127 resulted in its slower release in both acidic and basic media compared to free vitamin [163].

Digested kenaf (*Hibiscus cannabinus* L.) seed O/W NEs stabilized by a SCas, Tween 20, and β-CD complex demonstrated good lipid digestion, significant bioaccessibility of antioxidants (tocopherols and total phenolic contents), and lower phytosterol degradation rate compared to digested bulk oil, which indicates the possibility of their future application in food and nutraceutical industries [164].

#### **4. Other Functional Applications of Human Supplements**

This chapter is focused on all other applications of DISs and FSMPs except antioxidants, i.e., supplements affecting intestine, various nutraceuticals with beneficial effect against tumor cells or constituting nutritional support therapy in the treatment of cancer, supplements supporting mental and psychomotor development, supplements for prophylaxis of metabolic syndrome, supplements for osteoporosis management, and supplements against iron deficiency.

#### *4.1. Supplements Affecting Intestine and Absorption*

It is indisputable that for proper functioning of the intestines, nutrient absorption and prevention of malnutrition, it is necessary to have a suitable composition of the "good" intestinal microflora. Different diseases and subsequent treatments may change the composition of the intestinal microflora, which may result in various, initially intestinal problems. These problems can be avoided by using different products. Prebiotics are compounds in food that induce the growth or the activity of beneficial microorganisms. Probiotics are live microorganisms (in general, bacteria of the genera Lactobacillus and Bifidobacterium) intended to provide health benefits when consumed, generally by improving or restoring the gu<sup>t</sup> flora. Synbiotics refer to food ingredients or DISs combining probiotics and prebiotics in the form of synergism [165,166].

Probiotics, prebiotics, and synbiotics could suppress enteric pathogens, because they can compete with pathogenic microbiota for adhesion sites, inhibit the growth of pathogens, or stimulate, modulate, and regulate the immune response of the host by initiating the activation of specific genes in and outside the intestinal tract. Moreover, it was also shown that probiotics regulate fat storage and stimulate intestinal angiogenesis [167]. Probiotic encapsulation technology was developed rapidly in the past decade. Based on this technology, a wide range of microorganisms have been immobilized within semipermeable and biocompatible materials that modulate the delivery of cells [168].

Co-encapsulation of probiotic strains *Staphylococcus succinus*(MAbB4) and *Enterococcus fecium* (FIdM3) in alginate (2 g/100 mL) resulted in a significant improvement ( *P* < 0.05) in the survival of co-encapsulated cells when exposed to acidic (pH 2.0–3.0) and bile (0.3, 0.6 and 0.8 g/100 mL) conditions. Viability was maintained throughout the storage period and ranged from 8.1 log cfu/mL (Colony Forming Unit) to 7.9 log cfu/mL for about a period of 30 days at 4 ◦C [169].

Dietary factors such as prebiotics (e.g., inulin) play important roles in the growth of intestinal microbiota and may impact the intestinal health [170]. The encapsulation of the probiotic *Pediococcus pentosaceus* Li05 in alginate-gelatin microgels loaded with MgO NPs enhanced its viability by filling pores inside the microgels and thus, the ability of O2 and H+ ions to access the probiotic could be inhibited due to the neutralization of H+ ions in the gastric fluids by MgO NPs, thereby suppressing its acid-induced degradation. Such formulation could be considered as an appropriate DES for improving the efficacy of orally administered probiotics [171]. The effect of a prebiotic matrix consisting of inulin in concentrations 0%, 5%, 10%, 15% and 20% ( *w*/*v*) in alginate beads on the viability of encapsulated probiotic strains *Pediocucus acidilactici*, *Lactobacillus reuteri*, and *Lactobacillus salivarius* was investigated by Atia et al., and the researchers found that the beads with 5% *w*/*v* inulin were the most effective in bacterial protection against bile-salts and acidity [172].

Probiotics are also affected by prebiotics apart from other things. Therefore, Peredo et al. [173] investigated the influence of natural prebiotics potato starch, *Plantago psyllium*, and inulin co-encapsulated with alginate on the viability of *Lactobacillus casei* Shirota and two strains of *Lactobacillus plantarum* Lp33 and Lp17. The results showed a higher encapsulation yield when *P. psyllium* (94% for Lp17) and inulin (78% in Lp33) were used; *P. psyllium* ensured a higher viability of the bacteria during storage at 4 ◦C and the best protection in GI conditions.

Recent findings related to the production of probiotics, prebiotics, and nutraceuticals using a nanotechnology approach with respect to the functional foods was presented by Mishra et al. [174].

#### *4.2. Anticancer Nutraceuticals*

Nutraceuticals, such as soya bean, garlic, ginger, green tea, propolis, honey, RES, Q, EGCG, etc., may have chemopreventive effects. They are able to induce the apoptosis of cancer cells. These special foods can be used for chemoprevention or as a supportive therapy at treatment of tumor by standard anticancer chemotherapeutics [175–180].

In a review paper, McClements and Xiao [181] focused their attention on some most important anticancer nutraceuticals found in foods, the main factors affecting their bioaccessibility, absorption, and transformation, and different types of DESs and excipient systems improving the overall bioavailability of anticancer nutraceuticals.

(-)-Epigallocatechin-3-gallate is the most abundant catechin and also the most effective cancer chemopreventive polyphenol in green tea. This EGCG pronouncedly inhibited β-Car degradation in both MCT and corn O/W emulsions in a dose dependent manner and did not adversely affect lipid oxidation, while α-Lalb was not able to protect β-Car in MCT emulsions; their combination had a similar effect as EGCG alone [182]. A comprehensive review related to findings concerning the encapsulation of EGCG by means of nanocarriers was presented by Granja et al. [183].

Biopolymer core-shell NPs consisting of hydrophobic protein (zein) as the core and a hydrophilic polysaccharide (pectin) as the shell fortified with CUR showing strong anticancer activity with the diameter of 250 nm, which were converted into a powdered form resulting in good water dispersibility, were reported to be suitable for incorporating CUR into functional foods and beverages as DISs, and pharmaceuticals [184]. Bioavailable NEs loaded with nutraceuticals (CUR and fresh and dry tomato extracts rich in lycopene) with the hydrodynamic size of NEs approx. 100 nm applied in combination with doxorubicin enhanced cell viability in cardiomyoblasts (H9C2 cells) by 35–40% compared to that observed in cardiomyoblasts treated with doxorubicin alone, provided protection against oxidative stress, inhibited the release of IL-6, IL-8, IL-1, TNF-<sup>α</sup>, and nitric oxide by approx. 35–40%, and increased IL-10 production by 25–27% compared to cells without NE treatment. The best cardioprotective profile was showed by a lycopene-rich NE capable to effectively protect against doxorubicin-induced cardiotoxicity by reducing inflammation and lipid oxidative stress [185].

Both a cinnamon oil NE and a vitamin D encapsulated cinnamon oil NE with particle sizes 40.52 and 48.96 nm, respectively, arrested the cell cycle progression in the G0/G1 phase, showed an increased expression of Bax, capase3, and caspase-9, and decreased the expression of BcL2 proteins along with a considerable increase of apoptotic cell population and loss of mitochondrial membrane potential. The NE with cinnamon oil as a carrier for a lipophilic nutraceutical like vitamin D showing potential anticancer activity in human alveolar carcinoma cells could be also used in the food industry [186].

A nanonutraceutical formulation of ω-3 PUFAs (fish oil) could effectively inhibit the release of ROS and reactive nitrogen species from human neutrophils and murine macrophages, the production of the proinflammatory cytokines TNF-α and MCP1, and tumor-cell proliferation in FaDu head and neck squamous carcinoma and 4T1 breast cancer cells in in vitro cultures. The ω-liposomes, in which docosahexaenoic acid (DHA) was formulated, could be used for intravenous delivery of fish oil fatty acids resulting in beneficial effects in the treatment of inflammatory disorders and cancer [187]. It is known that DHA (ω-3 PUFA), a component of fish oil, suppresses rat mammary carcinogenesis, reduces cell growth, and induces apoptosis in human breast cancer cell lines. An acid stable liposome formulation of DHA with the use of ether and phytanyl lipids similar in structure to those found in Archaea having the mean particle size of 137 ± 12 nm and a slightly negative charge was resistant to oxidation and stable over the pH range of 1.0–7.4 at 37 ◦C for two hours. Cell viability in MCF-7 cells and apoptosis in both MCF-7 and MDA-MB-231 cells were reduced more effectively by this liposomal formulation than by free DHA, suggesting that it could be potentially used in breast cancer prevention [188].

The investigation of nanosized complexes prepared using high amylose corn starch and flax seed oil processed to powder of MPs by spray-drying and subsequently incorporated into bread formulation showed a considerable reduction of lipid oxidation in breads during baking due to the encapsulation as well as a decreased formation of carcinogen acrylamide, suggesting a beneficial effect of this nanoformulation on the final product quality and safety [189].

#### *4.3. Supplements Supporting Mental and Psychomotor Development*

Fermented soybean nanonutraceuticals administered to rats intoxicated with colchicine and showing impairment in learning and memory and decreased activity of acetylcholinesterase (AChE) caused an increase of AChE activity (42%), a reduced activity of GSH (42%), SOD (43%), and catalase

(41%), and decreased lipid peroxidation (28%) and protein carbonyl contents (30%), which suggests a possible neuroprotective efficiency of the nanonutraceuticals, and in addition, a significant amyloid-β and BACE-1 inhibition activity was demonstrated in an in silico study. The beneficial effect of the discussed nanonutraceuticals is associated with their strong antioxidant activity, and it could be assumed that they could also positively influence cognitive defects associated with Alzheimer's disease [190].

Encapsulation in bovine-milk exosomes could protect cargos against enzymatic and nonenzymatic degradation. RNAs encapsulated in exosomes could be delivered to circulating immune cells in humans, and some microRNAs and mRNAs in bovine-milk exosomes could regulate human gene expression and be translated into protein. Gene expression can be altered by low concentrations of dietary microRNAs through noncanonical pathways, such as the accumulation of exosomes in the immune cell microenvironment and microRNA binding to Toll-like receptors. In mice, the proliferation of intestinal cells was promoted by porcine-milk exosomes, suggesting that milk exosomes and their cargos could be used in human nutrition. Therefore, it was suggested that milk modified in this way could contribute to better mental, psychomotor, and functional development of infants [191].

Natural compounds that are commonly present in foods and beverages are regarded as promising molecules in a nutraceutical approach associated with life-long healthy diets. An increased attention is devoted to food molecules that are candidates to enter clinical trials as such or after targeted molecular engineering and could have a beneficial effect on amyloid neurodegenerative diseases. Natural phenols abundant in healthy food products, such as green tea, red berries, extra virgin olive oil, red wine, and spices, could be considered particularly promising [192]. Biodegradable poly(lactic-co-glycolic acid) NPs encapsulating ginsenoside Rg3 (an important constituent of ginseng, playing a significant role in memory and improving cognition) and thioflavin T, which showed neuroprotective effects, were reported to be a theranostic material for the detection and treatment of Alzheimer's disease [193].

#### *4.4. Supplements for Metabolic Syndrome Prophylaxis*

Sodium alginates could be used for the managemen<sup>t</sup> of GI tract disorders and the attenuation of components of the metabolic syndrome such as obesity, type 2 diabetes, hypertension, non-alcoholic fatty liver disease, and dyslipidemia. They could also protect cells during transplantation from immune responses of the host, and, in combination with antacid alginates, be applied in the treatment of gastric reflux disease. Moreover, alginates decrease food intake by inducing satiety, increase weight loss in patients on a calorie-restricted diet, and reduce both glucose and fatty acid uptake, and a decrease in blood pressure by alginates in rat models of hypertension was reported as well [194].

Using advanced proteomic and bioinformatic approaches, Kar et al. [195] characterized the protein components of six different protein sources (casein, partially delactosed whey powder, spray-dried porcine plasma, soybean meal, wheat gluten meal, and yellow meal worm) and predicted the bioactive properties of these protein sources after in silico digestion with monogastric proteolytic enzymes. The tested protein sources were potentially rich in bioactive peptides, in particular, angiotensin-converting enzyme inhibitors and peptides with antioxidant properties, and could be used as alternative sources of protein in animal feeds for monogastrics.

Temporal improvements in vitamin D status by vitamin D supplementation resulted in an increase in serum 25-hydroxyvitamin D concentrations and reduction of serum homocysteine concentrations suggesting that such treatment could reduce risk factors for CVD and may potentially contribute to the primary prevention of CVD [196]. Liposome-in-alginate beads were used to encapsulate the oyster hydrolysates showing antihypertensive effect to improve their bioavailability, protect them from degradation, and obtain sustained release; the release time of the oyster hydrolysate in the simulated GI fluid was up to 16 h [197]. Encapsulation of naringin, a flavonoid that occurs naturally in citrus fruits and possesses strong health benefits (recommended for the prevention of CVD and diabetes), in ternary NPs consisting of amylose, α-LLA, and β-Lglb resulted in a gradual release of naringin from

the ternary NP–naringin inclusion complex in simulated gastric and intestinal fluids, and ternary NPs effectively improved the bioavailability of bioflavonoid [198].

Mahmoud et al. [199] studied the impact of dietary camel whey protein administered as a supplement to streptozotocin (STZ)-induced diabetic pregnan<sup>t</sup> mice on the efficiency of the immune system of the offspring and verified its protective role in decreasing the tendency of the offspring to develop diabetes and related complications. A comparison of prophylactic effects of α-eleostearic acid rich nano and conventional bitter gourd seed oil emulsions in induced diabetic rats showed that the maximum efficiency in suppressing oxidative stress was achieved with a diet supplementation of 0.5% (*w*/*v*) NE with bioactive lipid-conjugated α-LNA, suggesting that such nanoformulation could be used as an appropriate nutraceutical against diabetes mellitus strongly attenuating an adverse impact of excessive ROS [200].

Although natural nanosized clinoptilolite and/or metformin did not affect pronouncedly the levels of serum glucose, minerals, and lipid profile in rats with high-fat-diet/STZ induced diabetes, the co-treatment of clinoptilolite with the drug notably increased high-density lipoprotein (HDL) cholesterol, while Cu and Ca levels increased only in the metformin group [201]. In STZ induced diabetic rats treated with nanosized clinoptilolite, blood glucose was found to decrease to near normal levels (12.4 vs. 27.5 mmol/L), but no significant impact on oxidative stress markers was estimated [202]. Nanosized clinoptilolite injected to STZ induced diabetic rats caused a partial improvement in their weight status and lack of undesirable effects, although beneficial changes in lipid profile were not detected, which could be connected with short study duration [203].

CS NPs loaded with *Stevia rebaudiana* leaf extract caused a considerable decrease of the mean fasting blood glucose level of treated diabetic rats in comparison with the diabetic control group, and serum levels of different enzymes and some antioxidants, e.g., catalase, reduced glutathione (GSH), and superoxide dismutase (SOD), were closer to normal levels in the group treated with NPs than in the control group [204].

By adding *Catathelasma ventricosum* polysaccharides (CVPs) to the redox system of selenite and ascorbic acid, spherical CVPs–selenite NPs with particle size approx. 50 nm were prepared, and based on serum profiles and antioxidant enzyme levels, it could be concluded that CVPs–selenite NPs showed a notably higher antidiabetic activity (*p* < 0.05) than other SeNPs, selenocysteine, and Na2SO3 [205].

To control the release of the anti-hyperglycemic agen<sup>t</sup> fisetin for nutraceutical and/or therapeutic applications, an oral controlled release system consisting of polymeric NPs (140–200 nm) based on poly-(ε-caprolactone) and poly(lactic-*co*-glycolic acid)-polyethylene glycol-COOH encapsulating fisetin was designed, which protected and preserved the release of the active compound in gastric simulated conditions, controlled the release in the intestinal medium, and showed an improved α-glucosidase inhibiting activity of fisetin compared to that of the commercial formulation acarbose [206].

NLCs loaded with betasitosterol, a phytosterol showing beneficial effects on reducing total cholesterol and low-density lipoprotein (LDL), with particle size 165 nm, zeta potential −13.5 mV, and EE 99.96%, which were incorporated in butter, showed good stability during three months' storage period and increased the antioxidant property of enriched butter during the storage period, suggesting the suitability of such nanoformulation for functional dairy products [207]. Multilayer CS–alginate–CUR NEs could be important for functional food development for combating obesity, because they increase satiety by retarding lipid digestion [143].

#### *4.5. Supplements for Osteoporosis Management*

Many supplements in pharmacies serve to prevent or mitigate the effects of osteoporosis. Calcium (Ca) has clearly been shown to have some positive effect on osteoporosis, although the bioavailability of Ca from classical preparations is approximately 10–15% [208–210]. Recently, a number of scientific teams investigated Ca supplementation by nano-Ca either as solid peroral DISs or as nano-Ca from the fortified milk. Experiments performed in vivo on ovariectomized (OVX) rats demonstrated much greater absorption (up to 89%) and overall bioavailability (up to 42%) of preparations with nano-CaCO3, citrate, or organically bound in shell oyster. Therefore, by in vivo studies it was confirmed that the application of nanosized Ca could improve Ca and even phosphorous content in bones [211–215].

Ca alginate NPs (200–500 nm in diameter) loaded with collagen peptide chelated Ca with the average diameter of approximately 150 nm and the Ca content of up to 130.4 g/kg notably enhanced Ca absorption and significantly increased femur bone mineral density and femur Ca content in rats, suggesting that they could prevent Ca deficiency and could be used as a new Ca supplement in the food industry [216]. A nanocomposite of whey protein hydrolysate chelated with Ca showed superb stability and absorbability under both acidic and basic conditions, which was beneficial for Ca absorption in the GI tract of the human body. Its pronouncedly higher Ca absorption on Caco-2 cells compared with Ca gluconate and CaCl2 in vitro suggested a possible increase in Ca bioavailability and thus its potential to be used as DIS for improving bone health of humans [217].

It is also important to remember that oral administration of Ca hydroxyapatite microcrystals can accelerate fracture healing and repair and even prevent osteoporosis [218]. Moreover, Zhang et al. [219] described the benefit of nanohydroxyapatite/CS composite for bone regeneration when it was administered by injection. In addition, these nanocomposites showed antistaphylococcal activity [220].

Additionally, CaCO3 from eggshell can be used as a Ca supplement [221]. Chicken eggshell powder became an attractive source of Ca for human nutrition. It can be added to food or drinks. For example, chocolate cakes were fortified by 3%, 6%, and 9% of them, and the results indicated that with respect to the Ca content, texture and sensory properties of the cakes, 6% eggshell supplementation (i.e., increased Ca content to 816.8 mg/100 g) was the best [222]. The preventive effects of nanopowdered eggshell (NPES) on postmenopausal osteoporosis in OVX rats was also studied, and the results were surprising. NPES fed rats showed an increase in bone mineral densities (BMD) by about 7% compared to OVX rats. Only powdered eggshell led to an increase of BMD by 2%. Serum analysis showed that NPES fed rats had a 22.4% higher osteocalcin level than OVX rats. Therefore, NPES attenuated the bone loss induced by ovariectomy in rats [223]. High-calcium yogur<sup>t</sup> as food for combat with osteoporosis was prepared using its fortification with 10-nm crystals of NPES. The addition of NPES up to 0.3% gave cow and buffalo's milk yogurts with acceptable composition, textural properties and sensory attributes, and this additive increased the Ca content of yogur<sup>t</sup> by about 15% [224].

#### *4.6. Supplements against Iron Deficiency*

Iron-deficiency anemia is the most common nutritional disorder worldwide with impact on health and economy. In spite of a number of commercially available supplements, this deficiency is a global public health problem due to the poor tolerability of the standard care soluble iron salts (such as ferrous sulfate), which results in non-compliance and ineffective correction of iron-deficiency anemia. On the other hand, poorly water-soluble compounds cause less sensory changes, but are not well absorbed [225]. Nanoformulations of iron were proposed to fortify food and feed to address these issues due to enhanced bioavailability, good product stability, limited side effects and the absence of changes of taste and color of the fortified foods [226]. In addition, in vitro and in vivo experiments have shown that iron NPs can be considered safe [227]. Ferritin, which is well absorbed [228] is itself composed of an iron oxide nanocore surrounded by a protein shell. Recently, Powell et al. [229] synthesized tartrate-modified, nano-disperse ferrihydrite having small primary particle size and enlarged or strained lattice structure (about 2.7 Å for the main Bragg peak versus 2.6 Å for synthetic ferrihydrite) that was able to efficiently provide GI delivery of soluble Fe(III) without the risk of free radical generation in murine models, where GI delivery did not depend on luminal Fe(III) reduction to Fe(II), and absorption was similar to that of FeSO4. This nanoformulation could be considered as a potentially side effect-free form of Fe supplementation to human suffering from anemia.

The most promising preparation (iron hydroxide adipate tartrate: IHAT) showed ~80% relative bioavailability to FeSO4 in humans and, in a rodent model, IHAT was equivalent to FeSO4 at repleting hemoglobin. Moreover, IHAT did not accumulate in the intestinal mucosa and, unlike

FeSO4, promoted a beneficial microbiota. In an in vitro study, IHAT was 14-fold less toxic than Fe(II) sulfate/ascorbate. The results of IHAT NPs observed from three-arm, double-blind, randomized, placebo-controlled trial conducted in Gambian children 6–35 months of age in relation to ferrous sulfate and non-inferiority in relation to placebo in terms of diarrhea incidence and prevalence confirmed the hypothesis that supplementation with IHAT eliminates iron deficiency and improves hemoglobin levels without inducing GI adverse effects [230,231].

Poorly water-soluble nanosized FePO4 with specific surface area approx. 190 m2/g made by scalable flame aerosol technology possesses in vivo iron bioavailability in rats comparable to FeSO4 and causes less color change in reactive food matrices than conventional iron fortificants. The addition of Zn or Mg oxides to nano FePO4 increases Fe absorption and also improves their color [232]. Additionally, Srinivasu et al. prepared nano ferric pyrophosphate (particle size 10–30 nm) as a potential food fortificant in iron-deficiency anemia and found that the peroral bioavailability of ferric pyrophosphate NPs in rats, calculated using hemoglobin regeneration efficiency, was 103.02% with respect to the reference salt, ferrous sulfate, while the NPs did not show any significant toxicity [233].

Salaheldin and Regheb biosynthesized biocompatible Fe3O4 NPs capped with vitamin C, and thus intestinal villi absorbed the NPs as vitamin C and not as an iron, because iron was coated with vitamin C. Clinical and histopathological studies on rats recommended the use of fortified biscuits with concentrations of 10 ppm and 30 ppm of nano iron; hemoglobin concentration increased from 9.9 ± 1.2 g/dL to 14.6 ± 1.1 and 16.7 ± 1.6 g/dL, respectively [234].

#### **5. Veterinary Nanoscale Nutraceuticals and Dietary Supplements**

As mentioned above for humans, nanoformulated DISs can also be applied for animals. In general, these veterinary DISs are regulated by the FDA's Center for Veterinary Medicine [235,236]. Nutraceuticals have become popular with the veterinary community; worldwide estimates of sales approach \$100 billion [237]. Therefore, many different products can be found for veterinary applications. For example, the use of clinoptilolite (natural zeolite comprising a microporous arrangemen<sup>t</sup> of silica and alumina tetrahedra) showing unique antibacterial properties as a DIS in food and unifying properties of an immunomodulator and nutraceutical could represent an alternative to antibiotic growth promoters in animals of veterinary importance. Valpotic et al. [238] focused their attention mainly on clinoptilolite potentials and limitations in cattle related to metabolic and endocrine status, oxidative stress, and systemic local inflammatory responses involved in reproductive and metabolic disorders of dairy cows.

Zinc (Zn), copper (Cu) and selenium (Se) are essential nutrients for animals and humans, because these metals occur in various metaloenzymes as co-factors [239]. Zn is a nutritionally indispensable trace element that is required for normal growth, bone development, feathering, appetite regulation, metabolic functioning of nearly 300 biochemical enzymes, hormone production, cell division, protein and DNA synthesis for all avian species [239,240]; so, it can affect animals production and reproduction performance [241]. Zinc deficiency in animals caused a decrease in feed intake, growth, serum insulin-like growth factor-I, and growth hormone (GH) and lowered the hepatic production of insulin-like growth factor-I, GH receptor, and GH binding protein [241–244]. In addition, Zn is used to decrease fermentation of digestible nutrients in intestines and improve nutrients digestibility and appetite. Dietary Zn supplementation stimulates feed intake probably caused by increased ghrelin secretion [245]. It was observed that it caused an increase in insulin-like growth factor expression in the small intestine mucosa [246]. Increased Zn concentration in the intestines influences their structure and function. The growth-stimulating properties of dietary Cu have been attributed to its antimicrobial action, however, it was shown that also intravenous injection of Cu to weanling piglets stimulated their growth [247]. It seems that the growth-promoting properties of high dietary concentrations of Cu complement its antimicrobial action [248].

Se is very important in animal nutrition, because it functions as an anti-oxidant assisted by vitamin E; e.g., Se is a cofactor of glutathione peroxidase (GSHpx), deiodinases, thioredoxin reductases, selenophosphate synthatase, selenoprotein P, selenoprotein W, etc. Se deficiency can be a major problem that can be reduced or prevented by supplementation with inorganic or organic sources of Se. On the other hand, Se in high concentration is toxic to human and animal [249]. Recent knowledge related to beneficial biological effects of SeNPs in the organism, absorption mechanisms, and nanotechnological applications for peroral administration were summarized by Hosnedlova et al. [250]. The applications of the above-mentioned nutrients (Zn, Cu, Se) in nanoscale formulations allow increased efficacy, enhanced absorption, lower overall doses, etc. [251,252]. In the following subchapters, an attention is mainly focused on the beneficial effect of some inorganic NPs (Zn, ZnO, Cu, CuO, Se, Ag) and nanoscale formulations containing organic active compounds (e.g., essential oils, vitamins) on growth performance and some important biochemical parameters of aquatic animals, poultry, pigs and other domestic animals like cattle, sheep, and rabbits. Nanoformulations have also found their way into the fortification of animal feeds [253,254].
