Potential of encapsulated phytochemicals in hydrogel particles

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11

Potential of encapsulated phytochemicals in hydrogel particles

1 ´ ˇ Nada Cujić Nikolić1, Katarina Savikin , Dubravka Bigović1, Kata Trifković2, Verica Ðorðević2 and Branko Bugarski2 1

Institute for Medicinal Plants Research, Belgrade, Serbia 2Department of Chemical Engineering, Faculty of Technology and Metallurgy, University of Belgrade, Belgrade, Serbia

11.1 INTRODUCTION Encapsulation process could increase stability of bioactive compounds during storage, protect them from harmful environmental conditions, extend shelf life, control the delivery, cover bitter taste of polyphenolics, and reduce the damaging effects of the gastrointestinal tract. Microencapsulation is a promising method to obtain particles that could stabilize bioactive compounds such polyphenols. On the other hand, nanoparticles are much smaller than cells, and could be directed to specific cell types, and due to their small size they are able to pass over the clearance system, allowing much longer circulation time. Hydrogel particles have attracted enormous attention as one of the most promising drug delivery systems due to their unique behavior via combining the characteristics of a hydrogel system (hydrophilic properties and huge water amount) with a very small size. In general, particle properties can be easily modified by varying the composition, shape, and size. Natural polymers such as alginate, chitosan, carrageenan, gelatin, xanthan, and other gums are the most extensively studied for preparation of hydrogel particles and synthetic ones such as polyvinyl alcohol (PVA), polyethylene oxide, polyethyleneimine, polyvinyl pyrolidone, and poly-N-isopropylacrylamide are widely used as biomaterials; these have shown excellent mechanical strength, biocompatibility, and nontoxicity. In recent years, extensive research has been carried out in designing and development of novel functional food ingredients with an aim of encapsulation technologies of polyphenols. Among dietary antioxidants, phenolic compounds are of special interest, due to their high content in plants, high dietary intake, and strong antioxidant activity. Hydrogel particles have been used as carriers for various polyphenol-rich plant extracts and isolated polyphenolic compounds proven to be powerful therapeutic agents and thus are used as promising food additives or pharmaceuticals.

Nanomaterials for Drug Delivery and Therapy. DOI: https://doi.org/10.1016/B978-0-12-816505-8.00009-6 © 2019 Elsevier Inc. All rights reserved.

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11.2 POLYPHENOLS AND THEIR BIOLOGICAL ACTIVITY Polyphenols represent secondary plant metabolites with unique chemical structure, containing at least one aromatic ring and many hydroxyl groups. They are the main compounds contributing to the antioxidant capacity of plant foods. These organic chemicals are characterized by the presence of large multiples of phenol structural units, from which they gained the term polyphenol in 1894 (Prasad, 2014). Phenolics are phytochemicals, which widely exist in numerous natural plants and foods, for example, herbs, fruits, and vegetables, thus are commonly used as part of the daily diet. In plants, polyphenols have function not only in protection against herbivores, infection, and UV radiation, but they also have function in attracting pollinators (Del Rio et al., 2010). To date, more than 8000 phenolics have been identified and they are usually classified as flavonoid and nonflavonoid compounds. The first group includes flavones, flavonols, isoflavones, flavanones, anthocyanidins, and flavan-3-ols, while the second group containing phenolic acids, stilbenes, and lignans. According to some researchers, average intake of polyphenols in Europe is around 1 g/day (Scalbert and Williamson, 2000; Zujko et al., 2012). Today, they are consumed as food supplements with health benefits. Intake of dietary antioxidants, as part of the optimal diet, is recommended to increase antioxidant defense of the body and prevent development of different chronic diseases (Arts and Hollman, 2005; Hooper et al., 2008). Potential health benefits of polyphenols are widely investigated and their beneficial effects are asserted not only in patients with cardiovascular risk factors, diabetes, metabolic syndrome, and dialysis, but also in healthy subjects. Numerous epidemiological studies on humans, as well as in vitro and in vivo studies on animals, have confirmed polyphenols’ health promoting effects. Polyphenols and their subgroups, anthocyanins, and procyanidins, represent one of the most potent natural antioxidants, and they exert positive impact on health and in diseases and states associated with marked level of oxidative stress. These compounds have beneficial effects on different chronic diseases but especially on cardiovascular disease, cancer cells proliferation, conditions caused by viruses and bacteria, and they could improve memory and digestion (Chrubasik et al., 2010). Polyphenols are considered as the most important dietary antioxidants, with antioxidant activity higher than vitamin C or E. They have strong protective effects against cellular oxidative stress and damage. Protective effects include free radicals scavenging and regulation of endogenous antioxidant enzymes activities (Rice-Evans et al., 1997; Scalbert et al., 2005; Mitjavila and Moreno, 2012). Antioxidant potential of plants depends not only on the total phenolic content, but also on the phenolic compounds structures. The content of polyphenols in plants varies and depends on the environmental conditions, habitat, cultivar, maturation, harvest time, and many other factors (Kulling and Rawel, 2008). Recent investigations suggest that polyphenols can protect biological membranes against lipid peroxidation by interacting with phospholipid bilayer. Uncontrolled lipid

11.2 Polyphenols and Their Biological Activity

peroxidation can be one of the main causes of cell damage leading to inflammation, cardiovascular damage, and other pathological processes. Oxidative damage of fatty acids could provoke changes in phospholipids’ structure and biophysical properties of cell membranes, such as fluidity (Rice-Evans et al., 1997). As it is previously mentioned, polyphenols can perform indirect antioxidant effects through the regulation of antioxidant enzyme activities (Scalbert et al., 2005). The suggested mechanisms include modulation of cell signaling pathways and gene expression. To date, different plant materials and plant parts have been analyzed as a potential dietary source of polyphenols. Among various plant foods, berries and especially chokeberry (Aronia melanocarpa) have a special interest due to their high content of polyphenols, which result in promising health effects. It is believed that berry fruits have the highest content of polyphenols, with wide range of phenolic subclasses, mainly flavonoids and phenolic acids (Oszmia´nski and Wojdylo, 2005). Due to substantial content of polyphenols, chokeberry is one of the strongest natural dietary antioxidants. The evaluation of the total phenolic content and antioxidant potential of different berry fruits detected significantly higher total antioxidant capacity (expressed as oxygen radical absorbance capacity) in chokeberry, compared with other berries, such as cranberry, blueberry, and lingonberry. Also, measurement of ability to scavenge DPPH [2,2-diphenyl-1picrylhydrazyl) and ABTS (2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid))] radicals confirmed high antioxidant activity of chokeberry (Oszmia´nski and Wojdylo, 2005). Except for chemically based assays, antioxidant activity of chokeberry is confirmed in in vitro and in vivo studies as well. Several human studies reported stimulation of red blood cells’ antioxidant enzymes after intake of chokeberry juice or extracts. Activities of erythrocytes’ antioxidant enzymes superoxide dismutase and glutathione peroxidase significantly increased after chokeberry juice consumption in metabolic syndrome subjects (Broncel et al., 2010). Chokeberry products have positive effects in activity of antioxidant enzymes not only in subjects with high cardiometabolic risk, but also in healthy subjects as well. The similar results were obtained in animal studies. Positive impact of chokeberry juice and extract consumption on blood pressure and other cardiovascular diseases is most probably related to its substantial content of phenolic compounds. As previously mentioned, polyphenols can affect hypertension and general cardiovascular health due to their ability to reduce vascular oxidative stress. The cardioprotective effects of polyphenols could also result from their impact on endothelial function. This is mostly referred to an increase in endothelium synthesis of nitric oxide (NO) and an improvement in flow-mediated dilatation. Phenolic compounds showed potential to increase NO level in vitro, accompanied with an enhanced activity of endothelial NO synthase (eNOS) (Appeldoorn et al., 2009). Among the numerous health effects of polyphenolic compounds, they have an important role in the regulation of lipid status by various mechanisms. Some authors suggest that phenolic plant extracts can reduce triglyceride levels, through their stimulatory effects on endothelium bound lipoprotein lipase, which

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hydrolyzes triglycerides into fatty acids (Devi and Sharma, 2004). Flavonoids can enhance activity of the enzyme lecithin-cholesterol acyl transferase, responsible for cholesterol removal from the bloodstream and which could cause the reduction in total and LDL cholesterol level (Katare et al., 2014). Some phenolic compounds, such as resveratrol, could reduce synthesis of triglycerides in the liver, by suppressing expression of genes encoding relevant enzymes (Gnoni and Paglialonga, 2009). Some studies reported that polyphenols can affect absorption of fatty acids and fats by changing the emulsification processes. Before the absorption, digestive products of dietary fats must be emulsified by bile acids and phospholipids in the intestinal lumen. By affecting emulsification of fats, polyphenols can decrease the activity of lipolytic enzymes and thus lower the fat absorption. It has also been suggested that polyphenols from green and black tea could increase the size of lipid droplets, formed in the emulsification process, and decrease the specific area of contact with enzymes. Increase in lipid droplets can result in smaller absorption of dietary fats (Shishikura et al., 2006). Positive impact on lipid status is important in terms of cardiovascular health also. Although hypertension is the most common cardiovascular risk predictor, only a small number of subjects have high blood pressure alone (Thomas et al., 2001). Dyslipidemia is, among others, a risk factor included in the total cardiovascular risk (D’Agostino et al., 2001). Consumption of polyphenols as dietary sources could also have effects on blood glucose level in subjects with risk factors for cardiovascular disease. In subjects with mild hypercholesterolemia, polyphenols intake could induce significant decrease in fasting glucose levels, while in patients with noninsulin-dependent diabetes mellitus, this effect could decrease levels of glycated hemoglobin (HbA1C) (Skoczynska et al., 2007; Simeonov et al., 2002). These effects on blood glucose indicate that polyphenols could have impact not only on initiation and progression of cardiovascular diseases, but also on metabolic disorders such as insulin resistance, metabolic syndrome, and diabetes. Some authors suggest that polyphenol-rich foods could have influence on insulin sensitivity, adipogenesis, and inflammation. It has been shown that different plant extracts rich with polyphenols could have beneficial effects on platelet function and their aggregation (Konić-Ristić et al., 2013a,b). Besides positive impact on cardiovascular health, many in vitro and human studies investigated potential beneficial effects of polyphenols on inflammation, neurological health, urinary tract, and cancer incidence. It is supposed that these effects are also associated with the high content of polyphenols and their activity. Inflammation, especially chronic, is associated with many conditions and diseases, such as adiposity, liver diseases, hypertension, and others. Numerous epidemiological studies investigated the relationship between cancer incidence and intake of fruits and vegetables rich with polyphenols, while many in vitro experiments indicate that polyphenols could affect tumor development and carcinogenesis. Some studies suggest that polyphenols may decrease the

11.2 Polyphenols and Their Biological Activity

risk of cancer based on the improvement of DNA oxidative damage. Berry fruits and their products have often been mentioned in many in vitro and in vivo as chemopreventive agents. Some authors suggested that chokeberry fruits and their extracts could have antiproliferative or protective effects against colon cancer (Kulling and Rawel, 2008). Chokeberry and its main derivates anthocyanins are suggested to have antimutagenic activity based on their radical scavenging properties, and also on the inhibition of enzymes involved in promutagen activation (Kokotkiewicz et al., 2010). Polyphenols have also been studied for their potential to improve neurological health, while it is believed that berries have impact on memory and learning. These beneficial effects are based on different mechanisms, including influencing on intracell neurological signaling and improving cerebrovascular blood flow (Del Rio et al., 2010). Beside these effects’ potential impact on cognitive functions, some authors have suggested that polyphenols could promote healthy aging. Beneficial effects of polyphenols on aging-related processes were investigated in numerous studies. For example, supplementation with chokeberry juice stopped changes in rats’ aortic wall and reduced the level of proatherogenic LDL cholesterol. This is also based on antioxidant activity because it is well known that free radicals and reactive species can provoke the age-related damage of the cells and tissues (Daskalova et al., 2015). Some findings also demonstrate reduction of anxiety and depression behaviors, as a result of polyphenols consumption. The researchers suggested that MAO-A/MAO-B inhibition could be a possible mechanism of this behavior (Tomić et al., 2016). Among numerous diseases, urinary infections are considered as one of the most persistent infections, requiring serious treatment with antibiotics or different antibacterial drugs. Long-term placebo controlled trial investigated effects of polyphenols-rich chokeberry juice consumption on incidence of urinary tract infections. Polyphenols consumption could reduce frequency of infections and use of antibiotics. Some authors suggested delayed bacteriostatic effects of phenolic compounds and their metabolites as a potential mechanism of action in urinary infection (Handeland et al., 2014).

11.2.1 DELIVERY OF PHENOLICS Basically, the delivery of phytochemicals is significantly determined by their physicochemical properties, for example, water solubility, partition coefficient, lipophilicity, and crystallinity. Many plant active compounds are poorly dissolved in oil or water (e.g., resveratrol, curcumin, quercetin, ellagic acid) and thus pose a problem to the route for their administration, transportation, and reaching the targeted places, resulting in a poor oral bioavailability. On the other hand, some phytochemicals have quite good solubility, but low intestinal permeability, for example, catechins, which again results in low bioavailability. According to Manach et al. (2005), more than 97 polyphenols, proanthocyanidins, galloylated

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tea catechins, curcumin, and the anthocyanins are the least absorbed polyphenols compounds. Polyphenols in general are recognized as bioactive compounds with low bioavailability and intensive metabolism, thus, their health effects should be attributed to their metabolites or compounds present in circulation after the consumption of polyphenol-rich foods. Additionally, identification of metabolites and their levels in circulation is required for the understanding of the polyphenols’ health effects. Recently, processes such as absorption, distribution, metabolism, and elimination of different phenolic subclasses have been increasingly investigated. Isoflavones, flavanones, and gallic acid are identified as the best absorbed polyphenolic compounds, but on the other hand procyanidins and anthocyanins are considered to be the ones with the lowest bioabsorption (Manach et al., 2005). In general, absorption of most phenolic compounds mainly takes place in the small intestine. Since phenolic compounds are mostly present in the form of glycosides in plants, absorption usually starts with cleavage of the sugar part and further diffusion of aglycones to enterocytes. Before polyphenols enter into the circulation, they can undergo the second phase of metabolism, which includes glucuronide, sulfate, or methyl derivates. Phenolic compounds that are resistant to hydrolytic enzymes pass unchanged to the colon, where they can be metabolized by bacterial microflora (Del Rio et al., 2010). Despite reports on low bioavailability of anthocyanins, polyphenols subclasses, recent studies have demonstrated their absorption and metabolism to a greater extent. Opposite to some earlier reports that anthocyanins can be found in blood only as glycosides, Kay et al. (2005) discovered the presence of conjugated metabolites in serum, after administration of cyanidin-3-glycosides in males. The cyanidin-3-glycoside, as the main anthocyanins compound, represented only 2% of the metabolites detected in the circulation. The other metabolites include conjugates of cyanidin-3-glucoside and degradation products, such as protocatechuic acid and phloroglucinaldehyde, phenylacetic, and phenylpropionic acids. Animal studies have also shown that anthocyanins can not only be rapidly absorbed in the gut and small intestine, but also that they have good tissue distribution (Talave´ra et al., 2004; Felgines et al., 2009). The anthocyanins were detected in different organs, including heart and adipose tissue, which confirms their potential beneficial effects on cardiovascular and metabolic diseases, as well as health promotion. In a study reported by Stanisavljević et al. (2015) chokeberry juice was subjected to in vitro gastric digestion in the presence of food matrix to determine the changes in polyphenols content and antioxidant and antiproliferative activity. Food addition decreased total phenolics and anthocyanins content, DPPH scavenging activity, and total reducing power for 36%, 90%, 45%, and 44%, respectively. The contents of individual anthocyanins were also affected by digestion and addition of complex food matrix, and their content decreased by approximately 90%. These results showed that a large proportion of phenolics undergo transformation during digestion. Combination of phenolic compounds and food matrix and prolonged exposure to mild acid environment during intestinal digestion could allow the transport of phenolic compounds to the colon.

11.2 Polyphenols and Their Biological Activity

11.2.2 ENCAPSULATION OF POLYPHENOLS As previously mentioned, plant polyphenols and their products, juices, and extracts are useful in prevention and treatment of many different chronic diseases, and the market demand for polyphenol-based products has grown steadily in the last decade (Galvan D’Alessandro et al., 2012). One of the most important reasons for encapsulation of phenolics and other phytochemicals is enhancing their bioavailability by changing the pharmacokinetics and biodistribution. On the other hand, many natural antioxidants are not stable at higher temperatures and alkaline pH, which later could provoke loss of their activity, for example, polyphenols are highly liable to epimerization (Kim et al., 2007). Further, some antioxidants are sensitive to humidity and UV light, such as thymol, cinnamaldehyde, caffeic acid, and carvacrol. Polyphenols have in general very astringent and bitter taste, which also could limit their use as nutraceuticals or their incorporation in food items. Today it is well known that medical plants have therapeutic effects due to synergistic actions of polyphenolic compounds. Thus there is increasing interest in encapsulation of plant extracts rather than isolated compounds. On the other hand, plant extracts and other products do not provide storage stability of polyphenols and the amount of bioactive compounds could be affected as previously mentioned by the presence of oxygen, light, humidity, or other inappropriate storage conditions (Bakowska-Barczak and Kolodziejczyk, 2011). In addition, several attempts have been made to increase the therapeutic potential of nutraceuticals by using bioavailability enhancers, coencapsulating the two sorts of nutraceuticals with synergistic actions into one delivery system (Aditya et al., 2013, 2015; Belˇscˇ ak-Cvitanović et al., 2011). Another reason for adding plant extracts to food products is prolongation of their storage shelf life and potential for preventing food diseases (Ferna´ndez-Lo´pez ¨ zvural et al., 2016). Namely, synthetic additives have been widely et al., 2005; O used in some food products (e.g., meat products) to inhibit the process of lipid oxidation and microbial growth. Due to the growing interest among consumers in such chemical additives, compounds obtained from natural sources (e.g., spices, fruit, vegetables, grains, oilseeds, herbs) are a preferred alternative compared with synthetic additives. Adding them in the form of micro- and nanoscale encapsulates can be also used as a means of control over food molecules and a strategy for modification of many macroscale characteristics, such as sensory attributes (texture, taste) and processability. More importantly, reducing the size of the encapsulates offers opportunities related with prolonged gastrointestinal retention time caused by bioadhesive improvements in the mucus covering the intestinal epithelium (Huang et al., 2010). Food grade or “generally recognized as safe” materials such as plant polysaccharides (e.g., starch, pectin, gum arabic, alginate, chitosan, carrageenan) or microbial origin (e.g., xanthan gum, dextran), food proteins (e.g., soy proteins, casein, gelatin, oat proteins, whey proteins), emulsifiers (e.g., lecithin), Tweens, Spans, sugar esters, and monoglycerides could be excellent solutions for delivery of phenolics and their use as food additives. Among

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synthetic polymers, methacrylic acid, methyl methacrylate and ethyl acrylate in the form of copolymeric commercial products (Eudragit), having mucoadhesive properties and being insoluble at low pH (enteric property) could be used for jejunum, duodenum, and colon delivery of various active compounds for pharmaceutical and food applications. Polyesters, such as PLGA, have been approved by the US Food and Drug Administration for ingestion and they are also popular due to biodegradability, biocompatibility, and versatile degradation kinetics. Although many different systems are now available to deliver bioactive components in functional foods, there is not much in vitro or in vivo evidence of their biological efficacies. Furthermore, examples of implementation of these delivery systems in real food products are also limited. Some recent examples of delivery systems for plant phenolics (plant extracts and pure compounds) and their applications in food and beverage products are listed in Table 11.1.

11.3 MICRO- AND NANOSYSTEMS FOR ENCAPSULATION OF POLYPHENOLS Particles with reduced sizes, microparticles, and especially nanoparticles, have an enormous impact on biomedical applications, which include drug delivery, imaging, and basic research. Miniaturization of therapeutic systems to the micron (1 1000 μm), submicron (100 1000 nm), and nanometer (1 100 nm) scales enabled biomedical application of therapeutic biomolecules to improve clinical efficiency (George et al., 2005). Among numerous data related to microparticles and nanoparticles, a clear universal boundary between nano- and microsize does not exist in the literature yet. There is a general rule based on expert convictions in the micro- and nanosciences that particle size between 1 and 100 nm is the optimum nanoscale range (Ferrari, 2005). Depending on the future application, any miniaturized system could be designed. Beside particle size, shape is another critical parameter that could significantly modify the function of micro- and nanoparticles (Champion et al., 2007). Particle shape could have an impact on uptake by the organism and release of active biomolecules. A variety of materials also have impact influence, mostly for targeting and delivery of therapeutic biomolecules (Aukunuru et al., 2003; Kim et al., 2004). Microparticles and nanoparticles have been an active biomedical research area due to their unique advantages: small sizes (excellent candidates for drug delivery), high surfaces, and possibility for targeting to specific cells, tissues, or organs. Nanoparticles are generally acknowledged as particles with dimensions less than B100 nm. They may be composed of any material. Nanoparticles have generated significant interest as biomaterials, primarily because of their unique size. From a physical perspective, nanoparticles are small enough to target the wanted cells. Beside these direct size benefits, nanoparticles are also interesting

Table 11.1 Examples of Delivery Systems for Plant Phenolics Benefits of Encapsulation

Food Application

Benefits in Food

6.7 7.6 μm particles

/

Hamburger patties

Özvural et al. (2016)

High shear homogenization

Micelles

/

Polysorbate 80 and soy lecithin

Microemulsification

Solid lipid nanoparticles 140 nm EE 75% 84%

/

/

Kakkar et al. (2011)

Solid lipids cetyl palmitate and glyceryl stearate 1 vegetable oils: grape seed oil, Hypericum perforatum oil and sea buckthorn oil

Modified high shear homogenization technique

Nanostructured lipid carriers 195 257 nm

• Increased solubility by about 1670folds • Enhanced in vitro anticancer activity • In vitro release prolonged up to 7 days • Enhanced in vivo oral bioavailability • Physical stability • In vitro antioxidant and antibacterial activity

Reduced lipid oxidation and decreased number of bacteria during 8 days storage at 4 C /

/

/

Manea et al. (2014)

Phenolics

Carrier Material

Technique

Properties

Green tea extract

Chitosan

In situ chitosan cross-linking with TPP followed by ultrasonication

Curcumin

Hydrophobically modified starch

Curcumin

Green tea extract

References

Yu and Huang (2010)

(Continued)

Table 11.1 Examples of Delivery Systems for Plant Phenolics Continued Phenolics

Carrier Material

Technique

Properties

Green tea extract

Maltodextrin, β-cyclodextrin and combination of both

Freeze drying (FD) Spray drying (SD)

Green tea catechins

Oil-in-water nanoemulsion stabilized with soy protein isolate

/

8 50 μm for FD 100 140 μm for SD Nanoemulsion 240 270 nm

Tea catechins

Chitosan and poly (γ-glutamic acid)

Nanoparticles 140 nm

EGCG

Chitosan hydrochloride and sulfobutyl etherβ-cyclodextrin sodium Caseinophosphopeptides and chitosan

Polyelectrolyte self-assembly method Inclusion complexation Ionic gelation

Nanocomplexes 150 nm

EGCG

Nanoparticles 100 200 nm

Benefits of Encapsulation

Food Application

Benefits in Food

/

Bread

Retained quality characteristics of bread along with the functionality

Pasrija et al. (2015)

• 2.78-fold increase in the bioaccessibility of catechins • Physically stable up to 20 days at 4C • increased permeability through Caco-2 monolayer Enhanced transport across Caco-2 monolayer /

/

/

Bhushani et al. (2016)

/

/

Tang et al. (2013)

/

/

Liu et al. (2016)

• Absence of in vitro cellular toxicity to Caco2 cell • Enhanced intestinal permeability and absorption of EGCG

/

/

Hu et al. (2012a,b)

References

Garcinia fruit extract

Whey protein isolate, maltodextrin and combination of both

Freeze drying (FD) Spray drying (SD)

Microparticles 15 100 μm for FD

/

Bread

Ezhilarasi et al. (2013, 2014)

/

Enhanced qualitative characteristics: higher ( )-hydroxycitric acid concentration, higher volume, softer crumb texture, desirable sensory attributes • Reduction of cooking loss of pasta /

Garcinia fruit extract

Whey protein isolate

Spray drying

/

/

Pasta

Hypericum perforatum extract

β-Cyclodextrin

Inclusion complexation by freeze drying

/

Tea polyphenol (TP)-Zn complex

β-Chitosan

Ionic gelation with tripolyphosphate (TPP)

Nanoparticles 85 nm EE 97%

Tea polyphenols

Carboxymethylchitosan and chitosan hydrochloride

Ionic gelation

Nanoparticles 407 nm

• Thermal stability at temperatures where the free extract was oxidized • Sustained release of TP-Zn complex over 5.5 h • Sustained release of polyphenols in PBS • Antitumor activity toward HepG2 cancer cells

/

/

Zhang and Zhao (2015)

/

/

Liang et al. (2011)

Pillai et al. (2012) Kalogeropoulos et al. (2010)

(Continued)

Table 11.1 Examples of Delivery Systems for Plant Phenolics Continued Phenolics

Carrier Material

Technique

Properties

Eugenol

Chitosan

Emulsion ionic gelation with TPP

Carvacrol

Chitosan

Oil-in-water emulsion followed by ionic gelation of chitosan with pentasodium TPP

Nanoparticles , 100 nm LC 12% EE 20% Nanoparticles 40 80 nm LC 3% 21% EE 14% 31%

Curcumin

Oil phase of emulsions: Corn oil (LCT), Miglyol 812 (MCT), Tributyrin (SCT)

Oil-in-water conventional emulsions and nanoemulsions

Quercitrin

PLA

Solvent evaporation method

Nanoemulsions (r , 100 nm), conventional emulsions (r . 100 nm) Nanoparticles 250 nm EE 40%

EGCG

Eudragit S100

Emulsion solvent diffusion method

Microparticles 16 μm

Curcumin

PLGA

Nanoparticles 160 nm EE 47%

Curumin 1 catechin

(W/O/W) double emulsion

High-pressure emulsification followed by solvent evaporation Two-step emulsification method

d43 3 μm EE 88% 97%

Benefits of Encapsulation

Food Application

Benefits in Food

• Improved thermal stability upon extrusion at 155 C

/

/

Woranuch and Yoksan (2013)

• Antimicrobial activity against Staphylococcus aureus, Bacillus cereus, and Escherichia coli Bioaccessibility of curcumin in the order MCT . LCT .. SCT

/

/

Keawchaoon and Yoksan (2011)

/

/

Ahmed et al. (2012)

• Sustained release over 14 days in physiological conditions Bioadhesive property in small intestine of rats 22-fold increase in oral bioavailability

/

/

Kumari et al. (2011)

/

/

Onoue et al. (2011)

/

/

Tsai et al. (2011)

4-fold increase in bioaccessability

/

/

Aditya et al. (2015)

References

Raspberry leaf extract 1 vitamin C hawthorn extract 1 vitamin C Ground ivy extract 1 vitamin C Yarrow, nettle extract 1 vitamin C olive leaf extract 1 vitamin C

Alginate and chitosan

Electrostatic extrusion

Beads D0.5 780 1780 μm EE 80% 89%

Polyphenolic profile of herbal microcapsules: phenolic acids, flavan-3-oils and flavons

/

/

ˇ BelšcakCvitanović et al. (2011)

Thyme extract

Alginate Alginate- Sucrose Alginate inulin

Electrostatic extrusion

Beads 730 μm EE 50% 80%

/

/

Stojanović et al. (2012)

EGCG

Liposomes-in-alginate Liposomes-in-chitosan

Vibrating nozzle extrusion

Microbeads 220 270 μm EE 97%

Fruit nectar

Liposomes-in-alginate/ chitosan Liposomes-in-alginate/ sucrose

Electrostatic extrusion

/

Thyme extract

Chitosan

Inverse emulsion polymerization

Microbeads (d50) 380 475 μm EE% 85% 91% Beads 220 790 μm EE up to 67%

6% 13% degradation of EGCG versus 33% of free EGCG /

Istenicˇ et al. (2016)

Resveratrol

Release of polyphenols above 220 C upon heating Stability at pH 6 for 2 weeks versus 70% degradation of free EGCG Prolonged release of resveratrol up to 24 h

/

/

Trifković et al. (2014, 2015)

Curcumin and Genistein

Glycerol monostearate 1 oleic acid 1 lecithin

Emulsification (high-speed homogenizer and ultrasonication)

/

/

Aditya et al. (2013)

NLC 108 122 nm

Prolonged release of polyphenolic compounds in simulated gastrointestinal conditions up to 6h Increased solubility (75% versus 20%) of active compounds antiprostate cancer activity

Balancˇ et al. (2016a,b)

EGCG, (2)-Epigallocatechin-3-gallate; PLGA, poly lactic-co-glycolic acid; TPP, tripolyphosphate; LC, loading capacity (mass of active compound per mass of sample, %); EE, encapsulation efficiency (mass of loaded active compound per mass of initial active compound, %); MCT, medium chain triacylglycerols; LCT, long chain triacylglycerols; SCT, short chain triacylglycerols; NLC, nanostructured lipid carriers.

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biomaterials because of their unique, size-dependent physical properties. Due to these interesting properties, nanoparticles have found application as carriers for drug delivery and as therapeutics.

11.3.1 MATERIALS USED FOR MICROPARTICLE AND NANOPARTICLE SYNTHESIS The choice of biomaterial for micro- and nanoparticle synthesis is dependent on the first order of application, duration of therapy, nature of the polymer (hydrophobic or hydrophilic, neutral or charged), and bioactive agents for delivering. The topics of current micro- and nanoparticles research are focused especially on drug delivery, controlled drug release, minimizing toxicity, and degradation of bioactive compounds (Dumitriu, 2002). Here we discuss some of the natural and synthetic materials used in micro- and nanoparticle preparations, but just a few are usually used as polymers in biotechnology.

11.3.1.1 Natural materials 11.3.1.1.1 Alginate Alginate is a copolymer of D-mannuronic and L-guluronic acid with β (1-4) linkages, and it is obtained from brown seaweed. This polymer has been widely investigated in pharmaceutical sciences due to its biocompatibility, low immunogenicity, and unique advantage of sol2gel transition in the presence of multivalent cations (Ca21, Mg21). The alginate micro- and nanoparticle preparation does not involve organic solvents or high temperature which can reduces the bioactivity of active biocompounds, for example polyphenols (Dumitriu, 2002). Nanoand microencapsulation of natural antioxidants and polyphenols has lately been the main focus in research systems and have been used extensively for protein, drug, and cell microencapsulation (Stojanović et al., 2012). Bioactives have been encapsulated in calcium chloride cross-linked alginate spheres with modified release (Wheatley et al., 1991), and polycations addition can reduce burst effect and enable sustained release (Wheatley et al., 1991). Alginate micro- and nanosystems have been studied from the beginning for nasal and oral delivery (Bowersock et al., 1996; Hari et al., 1996). Alginate miniaturized systems could also be prepared with other polymers such as chitosan. The addition of chitosan could modify characteristics of particles and release profile of bioactive compounds (Hari et al., 1996).

11.3.1.1.2 Chitosan Chitosan is a cationic polysaccharide with β (1-4) linkages, and it is obtained by alkaline chitin deacetylation. It is a polymer present in insect exoskeletons and marine crustaceans. Chitosan is a popular carrier for drug delivery of active compounds due to its biocompatibility, complete elimination from the body after degradation, and many primary amines as functional groups (Agnihotri et al., 2004).

11.3 Micro- and Nanosystems for Encapsulation of Polyphenols

Due to the presence of primary amines in structure, chitosan is soluble in aqueous acidic solutions, which could eliminate or reduce use of organic solvents during synthesis, and also offers many advantages for ionic encapsulation of negatively charged biomolecules (combinations of alginate and polyphenols). Particles obtained with chitosan could prolong mucosal residence time and increase mucosal permeability of bioactive compounds in case of nasal delivery (van der Lubben et al., 2001). Chitosan micro- and nanoparticles could also be used for oral drug delivery, which make them appropriate candidates if chitosan is used as a monocarrier or in combination with alginate (van der Lubben et al., 2001; Ahire et al., 2007).

11.3.1.1.3 Gelatin Gelatin is denatured form of animal-derived collagen, and demonstrates excellent biocompatibility and biodegradability. Particles obtained with gelatin have been investigated for sustained release applications (Ratcliffe et al., 1984; Tanaka et al., 1963). The first preparations made with gelatin were micropellets prepared by Tanaka et al. (1963). After that numerous molecules have been successfully encapsulated in gelatin such as different growth factors (Holland et al., 2007; Park et al., 2007, Pham et al., 2008; Patel et al., 2008b). Glutaraldehyde is a usual cross-linker for obtaining particles, and its concentration can modify release rate of biomolecules. For slow degradation, enhanced sustained release of drug, and enhanced in vivo biocompatibility, some researchers have utilized the naturally occurring cross-linker genipin to cross-link gelatin microparticles. The resulting particles had a reduced degradation rate and inflammation in vivo, in comparison with glutaraldehyde-cross-linked microparticles (Liang et al., 2003).

11.3.1.1.4 Dextran Dextran is a bifurcate glucose homopolysaccharide, and it is naturally synthesized from sucrose by some bacterial strains. Many biomedical studies have been used dextran as a carrier due to its biocompatible, biodegradable, easily derivatized properties. Dextran microparticles drug delivery depend on their biomedical applications (Chen et al., 2006; Cheung et al., 2005; Diwan et al., 2001; Demetriou et al., 1986; Zhang et al., 2008). Addition of different excipients in dextran could change loading efficiency and release of different bioactives.

11.3.1.1.5 Other natural materials for preparation of hydrogel particles Besides the previously mentioned carriers, other natural materials have also been used for micro- and nanoparticles synthesis. For example, collagen and hyaluronic acid have been used for delivery of different bioactive molecules (Berthold et al., 1998; Rossler et al., 1995; Swatschek et al., 2002; Kim et al., 2007; Lee et al., 2002). Natural materials as carriers are suitable for drug delivery applications because they are biodegradable and well-tolerated in the body.

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11.3.1.2 Synthetic polymers for preparation of hydrogel systems 11.3.1.2.1 Poly(lactic acid), poly(glycolic acid) and copolymers Bioresorbable aliphatic polyesters of lactic acid and glycolic acid are commonly used for controlled drug delivery (Lewis, 1990). Structurally, poly(lactic acid) (PLA) and poly(glycolic acid) are homopolymers and poly(lactic-co-glycolic acid) (PLGA) is a copolymer. Poly(d,l) lactic acid is not optically active, has amorphous nature, and it is mechanically weaker than l-PLA or d-PLA, which is an appropriate candidate for drug delivery applications. Particles made with PLGA were developed for the first time as carriers in the late 1970s (Tice and Lewis, 1980). From that time, these polymers have been synthesized in various morphologies, including microparticles, microneedles, pellets in different size, nanoparticles, films, and implants (Park et al., 2007; Siegel et al., 2006; Teixeira et al., 2005). The degradation rate of copolymers depends on several factors that relieve water availability to the ester linkages in the particles (Park, 1995; Shive and Anderson, 1997) such as crystallinity of copolymer. PLGA are more available for water penetrating compared with PLA linkages, which result in faster kinetics. Because of their excellent biocompatibility and biodegradation, high permeability, and nontoxicity, this polymer has been studied extensively for delivery of different biomolecules (Singh et al., 2008).

11.3.1.2.2 Poly(ε-caprolactone) PCL is a biodegradable, semicrystalline polymer with a glass transition temperature of B60 C. Polymerization of PCL was first studied in 1934 (Van Natta et al., 1934), but the biodegradable nature of the polymer was not recognized until the 1970s. Since then, numerous studies have investigated particles made with PCL for drug delivery applications because of its degradation characteristics, biocompatibility, high permeability with drugs, and deficiency of toxicity (Murthy, 1997). Their slow degradation and nonacidic degradation, compared with PLA/PLGA, make PCL particles appropriate for prolonged bioactives release. PCL could be combined well with other polymers, which results in modified permeability and degradation rates, and prolonged release profiles (Pitt et al., 1979).

11.3.1.2.3 Poly(ortho ester) Another class of biodegradable carriers developed especially for controlled drug delivery are the poly(ortho esters) (POEs). To date, the biocompatibility and biodegradability of POEs have been studied extensively. There are four different families of POEs. Degradation time can be shortened by modifying the polymer with lactic or glycolic acid segments. Varying the number of glycolic or lactic acid segments produces a range of degradation rates suitable for a wide range of delivery applications (Heller et al., 2002).

11.3 Micro- and Nanosystems for Encapsulation of Polyphenols

11.3.1.2.4 Other synthetic materials A number of synthetic materials have been examined for production of microand nanoparticles. For example, polyacrylamide particles were prepared almost three decades ago. These materials could also have been mixed with other natural or synthetic materials to achieve desired characteristics, such as varying degradation rate and release profile of bioactives.

11.3.2 PREPARATION OF BIOPOLYMER PARTICLES FOR DELIVERY OF PHENOLICS Biopolymer particles could be produced by different techniques and from a variety of polymers of natural origin. As described previously, micro- and nanoparticles could be prepared with synthetic monomers, natural polymers, and sometimes with semisynthetic polymers, using techniques that create polymer matrices of desirable sizes and structures. State-of-the-art techniques include different methods such as single and double emulsion solvent evaporation/ extraction methods, coacervation, spray drying, ionic gelation, and suspension cross-linking. Inclusion complexes of phytochemicals with cyclodextrins lead to their increased dissolution rate, membrane permeability, and bioavailability. Moreover, cyclodextrins improve the shelf life of food products and mask or reduce undesired smell or taste. Furthermore, encapsulation in β-CD improves the thermal stability of nutraceutical antioxidants. According to Kalogeropoulos et al. (2010) the inclusion complex between flavonoid-rich Hypericum perforatum (St. John’s wort) extract and β-cyclodextrin remained intact at temperatures where the free extract was oxidized. However, these systems often suffer from poor encapsulation efficiency of bioactives, which actually depends on ability for interactions and binding within the CD cavity. For example, encapsulation efficiencies for catechin, epicatechin, and quercetin from St. John’s wort extract were 27.5%, 30.0%, and 35.0% respectively (Kalogeropoulos et al., 2010). Microencapsulation in β-CD has been successfully applied in complex extract mixtures, like olive leaf extract (Mourtzinos et al., 2007), hibiscus anthocyanin-rich extract (Mourtzinos et al., 2008), propolis balsam (Kalogeropoulos et al., 2009) and Hypericum perforatum extract (Kalogeropoulos et al., 2010). Spray drying is a common technique used for encapsulation of a variety of bioactive compounds. Polyphenols have relatively high melting points ranging from 183 C (curcumin) to above 360 C (ellagic acid), but are susceptible to degradation on drying at higher temperature, and thus considered as heat labile compounds. This technique involves dispersion (emulsification) of aqueous drugs or direct addition of drugs in a polymer solution, which is then sprayed through a fine nozzle into a chamber (generally heated) where the solvent evaporates and particles are collected. The spray drying process lasts from a few milliseconds up to a few seconds, hence it can be successfully applied even for heat labile

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polyphenolic compounds (Ðorðević et al., 2015). However, lower outlet drying temperature and higher wall-to-core ratio are preferable parameters for preservation of thermosensitive compounds and their antioxidant activity (Pillai et al., 2012). Conventional particle fabrication methods like double emulsion and coacervation often have limitations, including particles aggregation as a result of high surface energies and mutual adhesion between particles, inefficient encapsulation, multistep processing, and residual organic solvents (Jain, 2000; Jalil and Nixon, 1990). Spray drying is a faster, more efficient process for encapsulating drugs and natural compounds in polymeric micro- and nanoparticles (Jain, 2000). This technique generally yields particles from 1 to 100 μm in diameter and encapsulation efficiency could be even 99%, depending on drug polymer interaction, solution viscosity, and nozzle characteristics, chamber temperature, flow rate and pressure, and cross-linker concentration (Jain, 2000; Wagenaar and Mu¨ller, 1994). Spray drying has been used for synthetic polymers such as PLGA, PLA, and PCL, and also for natural polymers including gelatin and polysaccharides as alginate and chitosan (Lorenzo-Lamosa et al., 1998). For polysaccharides (chitosan), the solution containing a mixture of polysaccharide, drug, and cross-linker is atomized similarly in a heated chamber. Cross-linkers such as glutaraldehyde forming an aldehyde amine could be used but cross-linker addition is not essential for particles formation. Produced particles might be lost due to agglomeration and adhesion to the chamber walls, and some research groups have developed a novel dual-nozzle system that simultaneously delivers both the polymer drug mixture and an antiadherent solution (e.g., mannitol) to prevent adhesion and aggregation (Takada et al., 1995). The low temperature spray drying method reported by Alkermes, Inc. (ProLease technology) was specifically developed to maintain integrity and activity of encapsulated biomolecules during the synthesis process. In this technique, powdered biomolecules and stabilizing excipients are suspended in a polymer solution in an organic solvent (acetone, ethyl acetate) and sprayed into a vessel containing liquid nitrogen followed by evaporation of the liquid nitrogen. The vessel also contains a frozen solvent such as ethanol for extraction of the polymer solvent from liquid nitrogen frozen droplets. With this technique could be produced particles with high encapsulation efficiency (95%) and sizes ranging from 50 to 60 μm (Johnson et al., 1997). Freeze drying can be considered as a favorable technique for protecting the thermolabile constituents such as polyphenols due to the very low temperature and oxygen-free environment. During the process water is being removed from a frozen sample by sublimation and desorption. This technique applies low temperature (240 C to 30 C) during the entire process, which includes three stages: freezing, primary drying, and secondary drying steps. Freezing involves formation of ice nuclei at below 0 C followed by the primary drying stage during which ice sublimes at low temperature and low pressure. During the secondary drying stage, temperature is slightly increased to cause evaporation of residual moisture present due to the bound and unfrozen water. Ezhilarasi et al. (2013) have shown up to 97% recovery upon encapsulation by freeze drying of ( )-hydroxycitric acid

11.3 Micro- and Nanosystems for Encapsulation of Polyphenols

(thermolabile compound) in comparison to 86% recovery when spray drying was applied (Ezhilarasi et al., 2013). Dripping/dispersing methods are techniques that include easy and reproducible procedures. This method is also known as ionic gelation, and this is a physiochemical technique where ionic polyelectrolytes (or charged polymers) are chelated with multivalent ions, producing cross-links. Ionic gelation is usually used with polysaccharides such as the sodium salt of alginic acid and chitosan, due to the simplicity and mild conditions of process (Bodmeier et al., 1989; Li et al., 2008; Park et al., 2004; Shu and Zhu, 2001). The ionic gelation process is a simple method and involves dropwise additions of polymer solution into the ion solution with dispersed drug or bioactive compound, with continuous stirring. By varying the preparation conditions, such as the ratio of polymer, cross-linking agent, concentration of polymer, stirring time, pH of the solution, the properties of biopolymer, the particle size, and the density of surface charge could be controlled and modulated to improve its encapsulation and release efficiency. The ionic gelation process can result in both nano- and microparticles ranging from several nanometers to 1000 μm. The process has been extensively applied for encapsulating drugs and different bioactives, and encapsulation efficiencies could range from 55% to 90% (Agnihotri et al., 2004; Ko et al., 2002). Very high encapsulation efficiency of antioxidants could be achieved (close to 100%), and very small particles (down to 100 nm). Belˇscˇ ak-Cvitanović et al. (2011) used the electrostatic extrusion technique for immobilization of polyphenol-rich extracts of various plants, such as thyme (Thymus serpyllum L.), nettle (Urtica dioica L.), hawthorn (Crategus laevigata), raspberry leaf (Rubus idaeus L.), olive leaf (Olea europaea L.), and yarrow (Achillea millefolium L.). These extracts were encapsulated in alginate chitosan microparticles, achieving a significant percent of encapsulation efficiency of polyphenols and preserved their antioxidant activity. Chemical stability and antioxidant activity was confirmed in study by Stojanović et al. (2012), where polyphenol-rich aqueous thyme extract (T. serpyllum L.) was successfully encapsulated in alginate inulin and alginate sucrose microbeads by electrostatic extrusion method. Emulsification methods could be used for the encapsulation of plant polyphenolic compounds and production of biopolymer particles, whereby emulsification procedure can be conducted as a classic polymerization in emulsion or as an inverse emulsion polymerization. Trifković et al. (2014, 2015) showed the potential of inverse emulsion polymerization for the encapsulation of thyme polyphenols within the chitosan particles. Biopolymer matrix can provide improved thermal stability of nutraceuticals. For example, Woranuch and Yoksan (2013) have shown 8-fold higher eugenol content and 2.7-fold greater radical scavenging activity of eugenol encapsulated in chitosan nanoparticles after extrusion at 155 C with a model plastic, that is, thermoplastic flour, than that containing free eugenol. Biopolymer matrix may sustain the release of polyphenols. For example, Zhang and Zhao (2015) synthesized β-chitosan nanoparticles, which prolonged release of tea polyphenol-Zn complex to 5.5 h. Furthermore, by careful selection

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of carrier material (mucoadhesive polymer) and optimizing the size of particles (nano), biopolymer particles can be designed to adhere and infiltrate into the mucus of the intestinal tract where they become unstable (gradually disintegrated) to allow the loaded polyphenols to be released and permeate through the paracellular pathway to the bloodstream. This concept was used by Tang et al. (2013) who developed nanoparticles from chitosan and an edible polypeptide [poly(γ-glutamic acid)]. They released about 40% of the loaded tea catechins in the simulated gastric fluid, but carrying the residual 60% of the loaded tea catechins they could be transported to duodenum (pH 6.0 6.6), jejunum and proximal ileum (pH 6.6 7.0), and distal ileum (pH 7.4) for release and absorption. Similarly, Trifković et al. (2015) showed the release of 50% 70% of loaded polyphenols from chitosan particles in simulated gastrointestinal conditions, whereas half the polyphenols amount was released in simulated gastric conditions and the other half in simulated intestinal conditions. Since the chitosan particles exhibit the properties of mucoadhesiveness and degradability in colonic environment, the remaining amount of polyphenols could be further released under conditions of large intestine for the prolonged periods of time.

11.3.2.1 Emulsion-based delivery systems for phenolics Emulsions contain continuous phase, dispersed phase, and emulsifier (surfactant), which acts as emulsion stabilizer. Conventional emulsions are usually in the range 1 100 μm while nanoemulsions are much smaller, 50 200 nm. Emulsions can be produced by a number of emulsification methods, categorized as high-energy emulsification methods (high shear homogenization, high-pressure homogenization, microfluidization, ultrasonic homogenization, and electrified coaxial liquid jets) and low-energy emulsification methods (phase inversion temperature method, colloidosomes, cubosomes, and microfluidic channels). Most of these methods are currently limited to laboratory use and have not been used in large batch production, which surely limits their application in food technology. In addition, production of emulsion often requires high oil or emulsifier contents, or the use of organic cosolvents. The emulsification solvent evaporation technique is one of the most commonly used in particle preparation, particularly for drug and bioactives encapsulation. The two-step technique calls for initial emulsification of polymer solution in an evaporative organic solvent, followed by internal phase solvent evaporation or extraction that results in hardening and precipitation of particles. Solvent evaporation is generally performed at atmospheric pressure (or sometimes under reduced pressure) to promote evaporation of the solvent. Commonly used solvents include ethanol, methylene chloride, ethyl acetate, and acetone/methanol mixtures. The single emulsion technique is generally employed for synthesis of particles encapsulating hydrophobic drugs. The polymer is dissolved in one phase (PLGA in oil phase or chitosan in aqueous phase), followed by addition of water to oil (W/O emulsion) or oil to water (O/W emulsion). To prevent coalescence of emulsion droplets, surfactants such as PVA could be used.

11.3 Micro- and Nanosystems for Encapsulation of Polyphenols

Emulsifying agents (surfactants) play an important role in emulsion selection for particles synthesis. If the emulsifier is oil soluble then it favors W/O type emulsion. Since the stability of the emulsion also depends on the difference in specific gravities between oil and water phases, W/O emulsions are sometimes difficult to create. Porosity of particles is controlled by rate of evaporation, ratio of internal aqueous phase, and viscosity of the polymer. Rapid solvent evaporation, as in the solvent extraction process, often results in porous particles as compared with the slow solvent evaporation process. The W/O/W double emulsion is a three-phase system in which polymer is dissolved in the oil phase with an internal aqueous phase, and immersed in an external aqueous phase containing surfactant or emulsifying agent. In a typical W/O/W double emulsion process, polymers (PLGA, PCL, PEG PLA copolymer) are dissolved in organic (oil phase) solvent followed by addition of a small volume (100 500 μL) of aqueous phase. This solution is subjected to strong homogenization or sonication to obtain the primary emulsion (Dorati et al., 2008). After that the primary emulsion is added to aqueous solution containing emulsifier (e.g., PVA) and homogenized. The organic solvent is eliminated by evaporation or extraction from W/ O/W double emulsion. Similarly, chitosan particles are usually prepared with prolonged W/O emulsification process where chitosan in the aqueous phase is emulsified in oil with surfactant, and then cross-linked using glutaraldehyde or ethyleneglycol diglycidyl ether to form hard droplets (Agnihotri et al., 2004). During the initial stage of the O/W emulsion, emulsion droplets are large because the aqueous phase is initially saturated with solvent, but when solvent evaporates, solvent concentration in the droplet decreases, causing a rapid shrinkage of droplet size and the formation of hardened particles. In the W/O/ W emulsion, different types of droplets could be formed, particles without aqueous phase entrapped, particles with only one aqueous phase, and a single droplet containing multiple particles. Therefore one has to find balance between possible benefits brought by the use of bioactives and potential side effects (e.g., cardiovascular diseases, obesity) caused by the use of high amount of lipids. These kinds of systems, especially nanoemulsions, provide bioavailability of the encapsulated phytochemicals. For example, Bhushani et al. (2016) have proved 2.78-fold increase in the bioaccessibility of major green tea catechins in simulated gastrointestinal conditions in comparison to nonencapsulated catechins. Namely, small emulsion droplets have large surface area, which increases the accessibility of different lipases and colipases and endogenous surfactants, such as bile salt, cholesterol, and phospholipids (Huang et al., 2010). However, digestion behavior of emulsions and thus bioaccessibility of encapsulated active compounds, depends on the type of lipids used for emulsion preparation. For this reason, in vitro lipolysis models (“pH-stat methods”) have been developed for quantifying the lipid digestion process and the release of substances into the colloid phases formed during lipid digestion. The fraction of a lipophilic component released into the mixed micelle phase after lipid digestion can be taken as a marker of its bioaccessibility. The bioactivity of

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a component, which is a measure of its specific biological affect, is determined by the fraction available for absorption. Such a research model has been used by Ahmed et al. (2012) who determined that under the simulated intestinal digestive environment, the length of the triacylglycerol chain (long, medium, or short chain triacylglycerols (LCT, MCT, and SCT), respectively) significantly influenced the initial digestion rate (SCT . MCT . LCT), final digestion extension of the lipid phase (MCT . SCT . LCT), and the bioaccessibility of encapsulated curcumin (MCT . LCT . SCT).

11.3.2.2 Precipitation and coacervation Coacervation usually uses the polymer physiochemical characteristics to form micro- and nanoparticles. Coacervation is a three-step process in which W/O emulsion is formed with polymer dissolved in the organic phase and drug dispersed in the aqueous phase. Stabilization of the coating occurs with solidification of particles in another organic nonsolvent followed by extensive washing, centrifugation, and freeze drying (Jain, 2000).

11.3.2.3 Micelle-based delivery systems for phenolics Micelles are formed by amphiphilic molecules, which have both hydrophilic and hydrophobic functional groups. The amphiphilic molecules (e.g., hydrophobically modified starch) above so-called critical micelle concentration in aqueous solution spontaneously create assemblies to form structured micelle complexes. The shell of micelles is bordered by hydrophilic region of the molecules, while hydrophobic region forms the cores, where lipophilic bioactives are entrapped. Solvent dialysis, solvent evaporation, coprecipitation and emulsification are known methods to encapsulate active compounds via micelles. Biopolymer micelles may prolong blood circulation time of phenolic compounds (Yu and Huang, 2010). Liposomes, colloid particles consisting of lipid bilayers, can be utilized for encapsulation of water-soluble, lipid-soluble, and amphiphilic bioactives, due to the presence of both lipid and aqueous phase within the liposome structure. Liposomes are submicron-sized spheres with an aqueous core and a bilayer membrane, where micelles consist of an aqueous core surrounded by a single layer of lipids. Liposomes are formed by one or several lipids, including sphingomyelin, phosphatidylcholine, or cholesterol. Liposomes could be classified as multilamellar (0.5 5 μm diameter), small unilamellar (B0.1 μm), or large unilamellar (0.2 0.8 μm) particles. Liposomes have excellent biocompatibility, and can encapsulate hydrophobic drug or bioactives between the lipophilic bilayers or hydrophilic in the core. The number of different production methods enables designing production procedures where high encapsulation efficiency can be achieved. For example, utilization of novel proliposome method for production of resveratrol loaded liposomes resulted in encapsulation efficiency of 97% (Isailović et al., 2013). Liposomes-in-hydrogel delivery systems are newly developed vehicles for the delivery of polyphenolic compounds. Since liposomes, and

11.4 Characterization of Micro- and Nanoparticles

lipid-based delivery systems in general, usually feature instability issues, the additional encapsulation of such systems within the hydrogel beads can be a tool to overpass the stability problems. In that respect, Balanˇc et al. (2016a,b) reported on encapsulation of resveratrol loaded liposomes in alginate chitosan and alginate sucrose microbeads, where high encapsulation efficiency were achieved, up to 91%. Produced complex systems supported the prolonged release of resveratrol (e.g., 70% of initially loaded polyphenols) up to 1260 min when compared with resveratrol release from nonencapsulated liposomes, where the release was conducted in the first 290 min. In addition, the successful encapsulation of resveratrol loaded liposomes within the hydrogel beads was confirmed by scanning electron microscopy (SEM) (Fig. 11.1).

11.3.2.4 Solid lipid nanoparticles A main advantage of solid lipid nanoparticles (SLNs) over polymeric particles is the fact that they are made from physiologically tolerated lipids, which decreases the potential for acute and chronic toxicities. SLNs combine the advantages of fat emulsions, polymeric nanoparticles, and liposomes. Thus they possess good physical stability (e.g., over the period of 1 year), high total drug content (up to 90%), and high entrapment efficiency (up to 85%). SLNs have been used as delivery systems for enhancing bioavailability of quercetin (Li et al., 2009), vinpocetine (Luo et al., 2006), and curcumin (Kakkar et al., 2011).

11.4 CHARACTERIZATION OF MICRO- AND NANOPARTICLES Behavior of particles depends on a number of factors. Surface charge and morphology, size, chemical composition, and tendency for aggregation play an important role and even minor replacements in any of these properties could result in significant changes. The usual methods for physiochemical characterization of micro- and nanoparticles are SEM, transmission electron microscopy, dynamic light scattering, Zeta potential measurement, X-ray photoelectron spectroscopy (XPS) analysis, atomic force microscopy (AFM), and optical or fluorescence microscopy. Microscopy techniques, including optical, fluorescence, transmission, and SEM are often used for studying particle surface morphology, size, and shape (Champion et al., 2007). It should be noted that electron microscopy techniques could involve preparation of obtained particles and prehandling steps, including dehydration or coating, which might provoke some changes in particle properties, especially hydrogel particles. For example, use of high vacuum might cause structural damage. For obtaining the size information and distribution or large number of particles, light scattering, optical microscopy, or some other appropriate method is preferred. The method that is used less frequently is AFM and this characterization method could also provide information about particle size, shape,

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FIGURE 11.1 Cross-sectional scanning electron microscopic (SEM) images of hydrogel microbead samples: ALG (A) ( 3 231.000), ALG-LIP/RES (B1) low magnification ( 3 14.000), with the insert showing flattening of a single liposome; ALG-LIP/RES (B2) high magnification ( 3 276.000), ALG/CH-LIP/RES ( 3 231.000) with the insert showing the surface membrane (C), ALG/SUC-LIP/RES (D) ( 3 26.700). ˇ This material is reproduced with kind permission of Elsevier, license number 4034800218654, from Balanc, ˇ B., Kalusević, A., Drvenica, I., Coelho, M.T., Djordjević, V., Alves, V.D., et al., 2016a. Calcium alginate inulin microbeads as carrier for aqueous carqueja extract. J. Food Sci. 81, 65 75; ˇ B., Trifković, K., Ðorðević, V., Marković, S., Pjanović, R., Nedović, V., et al., 2016b. Novel resveratrol Balanc, delivery systems based on alginate sucrose and alginate chitosan microbeads containing liposomes. Food Hydrocolloids 61, 832 842, Figure 3.

11.5 Influence of Encapsulation Process

morphology, elastic properties, particle adhesion, and deformation (Vakarelski et al., 2001; Tagit et al., 2008). XPS could provide information about the particle surface chemical composition (Xie et al., 2006) and usually XPS has been used to detect the presence of biomolecules or drugs on the particle surface (Xie et al., 2006; Chesko et al., 2008). Zeta potential measures surface charges, and this method is used to determine particle stability. A large value of zeta potential indicates stable particles that could form fewer aggregates. Zeta potentials can also be used for determining if particle surface is positively or negatively charged, which is important for particle modification. For example, cationic particles could be prepared by modifying PLGA particles with surfactants, with the surface positive charge confirmed by zeta potential. Besides these commonly used characterization techniques, differential scanning calorimetry has been used to assess changes in polymer thermal properties. Infrared spectra (IR or FTIR) have been used to analyze interactions between materials, carriers, and bioactive compounds and surface tension measurements have been used to evaluate particle aggregation.

11.5 INFLUENCE OF ENCAPSULATION PROCESS ON STABILITY OF POLYPHENOLS COMPOUNDS One of the methods to protect the beneficial properties of polyphenols and to overcome their problem with instability could be encapsulation technology. As previously mentioned, the main goals of encapsulation are enhancement of stability of bioactive compounds during storage, their protection from harmful environmental conditions, products’ shelf life extension, controlled delivery, covering their unpleasant taste (usually bitter), and circumventing the damaging effects of the gastrointestinal tract (Fang and Bhandari, 2010). Encapsulation method also represents a promising tool for bioavailability increment of polyphenols (Bakowska-Barczak and Kolodziejczyk, 2011; Belˇscˇ ak-Cvitanović et al., 2011). The aim of the next case study was to improve the functionality and stability of extracted chokeberry polyphenols by their encapsulation within calcium alginate matrix, as well as to explore the characteristics of the obtained system, which acts as a carrier with a prolonged/controlled release of active principles. To the best of our knowledge, before this study, literature data about encapsulation of chokeberry polyphenols were very limited. In a very few studies, chokeberry polyphenolic compounds were encapsulated via inclusion complexation (Howard et al., 2013) or stabilized via different drying methods. Horswald et al. (2013) investigated different drying methods such as spray, freeze, and vacuum drying, to preserve the chokeberry polyphenols. However, spray drying method requires processing with high temperatures, around 100 C and higher, when degradation of active compounds might occur, especially anthocyanins (Leong and Oey, 2012; Stojanović et al., 2012).

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11.5.1 ENCAPSULATION OF POLYPHENOL-RICH CHOKEBERRY EXTRACT WITHIN THE HYDROGEL BEADS PRODUCED BY MEANS OF ELECTROSTATIC EXTRUSION TECHNIQUE Еlectrostatic extrusion process was used for obtaining hydrogel microbeads with encapsulated chokeberry extract. As previously mentioned, chokeberry (A. melanocarpa) is a rich plant source of polyphenols with many confirmed health benefits. The effects of different carrier types (low and medium viscosity alginate), addition of inulin as filler, and the needle diameter (18, 20, 22 gauges) on the morphological characteristics, encapsulation efficiency and release properties of the hydrogel microbeads were studied. Among numerous microencapsulation methods, electrostatic extrusion stands as a simple, precise, efficient, and economical technique for obtaining particles with encapsulated bioactives. As already mentioned in Section 11.3.2, electrostatic extrusion is based on formation of charged stream of small particles using electrostatic forces that disrupt the liquid filament at the tip of a needle (Manojlović et al., 2008). This method has great potential for preparation of microbeads with controlled delivery and appropriate organoleptic and pharmacological characteristics and uniform size, which could be applied in the food and pharmaceutical industry (Pru¨sse et al., 2008). Among various carriers for encapsulation, sodium alginate has attracted great attention, due to its excellent biocompatibility; possibility to delay the release of active compounds, in these case polyphenols (due to the properties of its carboxyl groups); as well its biodegradability and nontoxicity (Stojanović et al., 2012). During the optimization of the microencapsulation process, besides different types of alginate as carriers, addition of inulin as a filler was also investigated to obtain the best loading and release profiles of polyphenols. Inulin also has a potential as functional food and pharmaceutical preparations as a source of dietary fibers. Inulin is soluble, natural polysaccharide with low calorific value and strong positive influence on the immune system due to its property to stimulate the intestinal microflora (Vlaseva et al., 2014; Beirao-da-Costa et al., 2013; Silva et al., 2004). Different factors such as bead size, viscosity of alginate carrier, and addition of inulin as a filler affected actual load and release of phenolics. Hydrogel microbeads had regular shape and size ranging from 800 to 1340 μm, depending on the type of carrier. As expected, the biggest needle diameter led to the formation of the biggest particles (1176.7 1267.7, 1123.3 1173.3, and 800 840 μm, for needle diameters 18, 20, and 22 gauges, respectively). Depending on the further application, different size of microbeads is desirable. Large particles provide more extended release of the encapsulated compounds, while smaller sized beads are preferable for better organoleptic characteristics of food or pharmaceutical products (Stojanović et al., 2012; Trifković et al., 2014). By optimization of process parameters, such as applied voltage, needle diameter, and viscosity of polymer solution, it is possible to obtain particles with uniform size of desired diameter (Stojanović et al., 2012; Belˇscˇ ak-Cvitanović et al., 2011). Obtained

11.5 Influence of Encapsulation Process

particles had spherical shape, and utilization of the electrostatic extrusion technique for the microbeads production and encapsulation of chokeberry extract resulted in the formation of particles of high uniformity. The addition of inulin affected the microbeads’ size. Particles prepared with inulin were larger than microbeads without inulin. The addition of inulin as a filler in the alginate solution caused the beads’ enlargement at some extent. High viscous alginate chokeberry solution, related to the high content of sugars in chokeberry extract (Kokotkiewicz et al., 2010; Kulling and Rawel, 2008), may be the explanation for the relatively big particles obtained. Particles obtained with medium viscosity alginate carrier (1.5% w/v), using inulin as filler (5% w/v) and medium needle size (20 gauges) showed the best results in the actual loading and release studies of encapsulated polyphenols. The loading efficiency depends on several factors including polymer hydrophilicity, porosity, cross-linking, and sometimes the most important, interaction between polymer and extract components (Stojanović et al., 2012; Trifković et al., 2014). The increase of alginate viscosity induced higher encapsulation efficiency, and the medium alginate had more concentrated network, created fewer pores, the surrounding membrane was thicker in comparison with low alginate. Porous structure of low alginate permitted facile diffuse of encapsulated compounds to an external solution. Greater polyphenols amount was presented in particles obtained with inulin than without. Polyphenols have higher affinity to alginate/inulin carrier compared with pure alginate and a possible explanation for this is interaction between polar parts of polyphenols groups and inulin hydroxyl groups (Balanˇc et al., 2016a,b). Similar methodology was previously described by Isteniˇc et al. (2015), who achieved the encapsulation efficiency of 24.5%, for resveratrol into calcium alginate submicron beads. This study explained that the loss of resveratrol was the consequence of diffusion through large specific area of alginate particles into the oil, phase with the same solubility as resveratrol. Different needle diameters provide different particle sizes of microbeads from which the polyphenols are released, and the best result was achieved with a needle of 20 gauges. The lowest polyphenols release was achieved with the smallest microbeads obtained with needle diameter of 22 gauges. Bigger microbeads allowed much slower release, which is appropriate in the case when prolonged delivery is preferable. Low viscosity alginate is suitable when the fast release of polyphenols is desired, but medium viscosity alginate is more favorable for prolonged release (Aizpurua-Olaizola et al., 2016). Comparing the results for phenolics release from the alginate and alginate/inulin particles in different environment (water or acidic), liberation of polyphenols was better in water than in an acidic environment. This could be important for the absorption and bioavailability in human organism indicating that microencapsulated chokeberry phenolics would rather be absorbed in the alkaline intestinal fluid than in gastric fluid. The explanation for this behavior probably derived from destruction of alginate hydrogel beads in acidic medium and swelling in neutral and alkaline medium, presumably of the presence of the D-mannuronic

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acid and L-glucuronic acid units. Large number of carboxyl groups are protonated in acidic solution, while in alkaline environment are ionized and refused, hydrogel particles swelled, and maximum water absorption and better release are achieved. Abd El-Ghaffar et al. (2012) published similar results with riboflavin alginate hydrogels which reduced size and shrank at low pH values. This pH sensitive characteristic makes alginate beads promising carriers for controlled release. Leick et al. (2011) demonstrated that anthocyanins at pH 3 are positively charged and alginate negatively charged due to the presence of carboxylic anions. A possible explanation is that some of anthocyanins and polyphenols polymerize, and interaction makes polyphenols’ release difficult and some part of them remains trapped in the particles. Most of the polyphenolic compounds from microbeads were released speedily and then reached a plateau. Release from chokeberry encapsulated hydrogel microbeads had a phase of rapid release in the first 10 min (burst effect), followed by a period with a constant release 10 20 min (lag time) and then a decrease of polyphenols content probably caused by saturation of dissolution medium. Release profile of encapsulated hydrogel microbeads was prolonged to 10 min. These results are in accordance with previous findings where polyphenolics, as water-soluble compounds, were released very rapidly from microbeads in the surrounding environment (Stojanović et al., 2012; Belˇscˇ ak-Cvitanović et al., 2011; Beirao-da-Costa et al., 2013). In the case of freeze dried particles made with inulin as filler, release was prolonged. Dried forms of microbeads usually have greater stability and mechanical stiffness compared with hydrogel forms thus they are preferable for utilization in foods and pharmaceuticals products (Stojanović et al., 2012). Dehydration that occurs during the drying process disturbs the structural integrity of the calcium alginate matrix, which consequently leads to reduction of microbeads’ sphericity. As dried forms of microbeads are preferable for utilization in foods and pharmaceuticals industry, effect of freeze drying was also investigated. After freeze drying process, particle size was reduced for 18% 24% comparing with hydrogel microbeads. Formulation prepared with addition of inulin showed the smallest size reduction, indicating that inulin has an impact on drying process. On the other hand, hydrogel microbeads contained 0.23 0.24 mg GAE/g, whereas freeze dried contained 3.24 3.57 mg GAE/g of encapsulated polyphenols, and release profile of encapsulated extracts was prolonged even to 40 min. Freeze dried particles had about 14 15 times higher amount of polyphenols compared with the hydrogel particles of the same type. These results showed that after drying, alginate or alginate/inulin beads became more concentrated with total phenolic entrapped. Similarly, Stojanović et al. (2012) reported the influence of drying method on the encapsulated alginate microbeads, and the highest amount of polyphenols were retained after freeze drying method (4.15 mg GAE/g), which was two times higher compared with hydrogel beads (2.04 mg GAE/g). Aizpurua-Olaizola et al. (2016) found freeze drying as the best solution to obtain fine granulated microbeads (30% polyphenols losses during drying)

11.6 Conclusion

compared with two different drying methods, air and heat dried at 50 C (more than 70% losses), where microbeads were stuck together, which is not appropriate for oral administration. SEM analysis was used to obtain information about surface morphology and shape of alginate and alginate/inulin microbeads (blank and with encapsulated extract), as well as to confirm the influence of inulin on these parameters. Microbeads’ characteristics such as shape and surface morphology can significantly affect the flow rate of particles, which could be extremely important for production of some pharmaceutical forms (i.e., conventional hard capsules). SEM micrographs confirmed that dried particles obtained by electrostatic extrusion were uniform in shapes, without destruction and damages, and that the addition of inulin as a filler improved the final properties of the microbeads. The SEM analysis showed that freeze drying could be used as a promising method for preservation of hydrogel beads. Presence of aggregates and particles of irregular shape was not noticed. This was one of the pertinent results in this study, because it is crucial for further industrial application. FTIR analysis was used to analyze the functional groups of the polymers and chokeberry extract, as well as to examine the possible interactions between chokeberry extract’s compounds and matrix. Spectra indicated that there was no chemical interaction between extract compounds and matrixes and extract was encapsulated without strain. FTIR analysis showed several relevant changes in the spectrum of alginate or alginate/inulin systems with the encapsulated chokeberry extract in comparison with blank microbeads, which confirmed that the extract was successfully incorporated into the particles. Due to the extended storage and stability, dry microbeads showed the best potential as a delivery system suitable for pharmaceutical or functional food industry. Drying process affected the encapsulation efficiency, the amount of encapsulated polyphenols increased in freeze dried beads, and the release profile of encapsulated extracts was prolonged to 40 min. The results of our study showed that chokeberry polyphenols could be successfully encapsulated in microparticles. Such microbeads seem to be a promising food additive and could be incorporated into dietary supplements, functional food or pharmaceutical and cosmetic preparations.

11.6 CONCLUSION Many previously mentioned studies showed and confirmed that hydrogel microand nanoparticles have gained considerable attention in recent years as one of the most promising drug delivery systems owing to their unique potential via combining the characteristics of hydrogel systems, hydrophilicity and extremely high water content. This is especially important for encapsulation of natural bioactive compounds such as polyphenols. Polyphenols have documented health beneficial

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effects, especially antioxidant potential. The problems with polyphenols are low stability, and sensitivity to oxygen, light, and humidity presence. New encapsulation techniques could be a promising way to overcome problems with polyphenol instability, low bioavailability, and degradation. Encapsulated polyphenols improved classic hydrogel systems leading to a promising strategy for protection and delivery of phenolics, keeping biological activity, and increasing the daily intake of antioxidants when they are implemented in food or pharmaceutical products.

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FURTHER READING Alsberg, E., Kong, H.J., Hirano, Y., Smith, M.K., Albeiruti, A., Mooney, D.J., 2003. Regulating bone formation via controlled scaffold degradation. J. Dent. Res. 82 (11), 903 908. ´ ˇ Cujić, N., Savikin, K., Janković, T., Pljevljakuˇsić, D., Zdunić, G., Ibrić, S., 2016a. Optimization of polyphenols extraction from dried chokeberry using maceration as traditional technique. Food Chem. 194, 135 142. ´ ˇ Cujić, N., Trifković, K., Bugarski, B., Ibrić, S., Pljevljakuˇsić, D., Savikin, K., 2016b. Chokeberry (Aronia melanocarpa L.) extract loaded in alginate andalginate/inulin system. Ind. Crops prod. 86, 120 131.