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الرئيسية Bioactive Molecules in Food
Bioactive Molecules in FoodJean-Michel Mérillon, Kishan Gopal Ramawat
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© Springer Nature Switzerland AG 2019 Jean-Michel Mérillon and Kishan Gopal Ramawat (eds.)Bioactive Molecules in FoodReference Series in Phytochemistryhttps://doi.org/10.1007/978-3-319-78030-6_23 74. Are Myristica fragrans L. (Myristicaceae) and Its Phytochemicals Useful for Human Health? Monica Rosa Loizzo1 , Vincenzo Sicari2 , Jianbo Xiao3, 4 and Rosa Tundis1 (1)Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, Rende, Cosenza, Italy (2)Department of Agricultural Science, Mediterranean University of Reggio Calabria, Reggio, Calabria, Italy (3)Institute of Chinese Medical Sciences, State Key Laboratory of Quality Research in Chinese Medicine, Macau University, Taipa, Macau (4)Institut für Pharmazie und Lebensmittelchemie, Universität Würzburg, Würzburg, Germany Monica Rosa Loizzo (Corresponding author) Email: firstname.lastname@example.com Vincenzo Sicari Email: firstname.lastname@example.com Jianbo Xiao Email: firstname.lastname@example.com Rosa Tundis Email: email@example.com Abstract From Myristica fragrans L. (Myristicaceae) two spices, nutmeg and mace, were obtained. This plant is traditionally used in China, India, and African countries for the treatment of several diseases, such as decreased appetite, diarrhea, muscle spasm, rheumatism, etc. Recently, M. fragrans has been investigated for antioxidant, anticonvulsant, analgesic, anti-inflammatory, antidiabetic, hypolipidemic and hypocholesterolemic, antibacterial, and antifungal potential. A perusal analysis of literature evidenced that M. fragrans deserves more attention by scientific community to explore its full range of bioactivity in the welfare of the society especially in emergent Countries. Keywords Myristica fragrans Chemical profileBioactivityToxicity List of Abbreviations 5-HT Serotonin AChE Acetylcholinesterase AMPK AMP-activated protein kinase BChE Butyrylcholinesterase DM Diabetes mellitus GABA γ-Aminobutyric acid IL Interleukin LDH-A Lactate dehydrogenase MCP Monocyte chemotactic protein MIP Macrophage inflammatory protein NO Nitric oxide PAS Peripheral anionic site THF Tetrahydrofuran mixture 1 Introduction Myristica fragrans L. (Myristicaceae) is an indigenous plant to India, Indonesia, and Sri Lanka. Now it is cultivated in many tropical countries of both hemispheres as well as in South Africa [1]. The tree is a small evergreen, not more than 40 feet in height, with smooth, greyish-brown bark, green on the younger branches. The alternate leaves are oblong-ovate, acute, entire, smooth, and dark-green. The flowers are very small and unisexual. The fruits, smooth and yellow, resemble a pear grooved by a longitudinal furrow and contain a single erect seed. The seed of the plant is known as "nutmeg" and the arillus of the seed is called "mace". Both kernel and arillus are rich in essential oil that imparts the characteristic aroma and taste to nutmeg as a unique culinary spice. Primary metabolites including carbohydrates, lipids/fatty acids, and proteins constitute up to 80% of the weight of dry nutmeg kernel while the remaining weight comprises secondary metabolites such as terpenes, phenolic acids, polyphenols, and pigments [2]. Since ancient times, M. fragrans nutmeg and its oil are used in Chinese and Indian traditional medicines for illnesses related to the digestive and nervous systems. Nutmeg is a rich source of essential oil, triterpenes, and various types of phenolic compounds that are responsible of the biological activities that may support its use in traditional medicine. In this chapter, we reported and critically discussed data from literature published from 2000 to 2017 on the M. fragrans as source of bioactive compounds. 2 Bioactivity of Extract and Isolated Compounds 2.1 Effects on Metabolic Syndrome Diabetes mellitus (DM) is a metabolic disorder characterized by hyperglycemia with impaired metabolism of carbohydrates, fat, and proteins due to defects in insulin secretion, insulin action, or both [3]. Spices are extensively used to enhance the taste and flavor of foods and are known to possess several medicinal properties including hypoglycemic effect. In this context, Somani and Singhai [4] reported that M. fragrans petroleum ether extract decreased blood glucose levels in normal, glucose fed, and alloxan induced diabetic rats by a mechanism of action involving a promotion of release of insulin from β-cells. Moreover, after oral administration a decrease in the rate of intestinal glucose absorption or potentiation of pancreatic secretions or increasing the glucose uptake was observed. The dose-dependent insulin secretion on isolated islets of Langerhans was confirmed by Patil et al. [5]. Moreover, this spice showed α-amylase inhibitory activity with an IC50 value of 0.85 mg/mL. A moderate hypoglycemic effect was confirmed for M. fragrans fruits ethanol extract in streptozotocin-induced diabetic rats [6]. Mace dried powered extract showed carbohydrate-hydrolyzing enzyme inhibitory activity with IC50 values of 62.1 and 75.7 μg/mL against α-amylase and α-glucosidase, respectively [7]. Among M. fragrans isolated constituents, macelignan (1) (Fig. 1) exerted a promising antidiabetic activity since it enhanced the insulin sensitivity and improved lipid metabolic disorders by activating peroxisome proliferator receptor-alpha/gamma and reducing endoplasmic reticulum stress [8].[image: ../images/438254_1_En_23_Chapter/438254_1_En_23_Fig1_HTML.png] Fig. 1Macelignan (1), myristicin (2) and malabaricone B (3) In addition, macelignan (1) possess an inhibitory action against advanced glycation end products, an effect that adds to its potential role in the management of diabetes and metabolic syndrome [9]. Frequently DM is associated to an impaired metabolism of fat. Ram et al. [10] showed that oral administration of nutmeg extract (500 mg/kg body weight) for 60 days to hyperlipidemic albino rabbits significantly reduced the lipoprotein lipids level. Several research studies evidenced that AMP-activated protein kinase (AMPK) activators mimic or potentiate the exercise-related effects. Therefore, the activation of AMPK could be used as drug target for the prevention and treatment of obesity and diabetes by regulating the glucose and lipid homeostasis [11]. Tetrahydrofuroguaiacin B, and nectandrin B at 5 μM produced strong AMPK stimulation in differentiated C2C12 cells. In addition, the preventive effect of a tetrahydrofuran mixture (THF) containing tetrahydrofuroguaiacin B, saucernetindiol, verrucosin, nectandrin B, nectandrin A, fragransin C1, and galbacin on weight gain in a diet-induced animal model was examined. Results evidenced that final body weights and weight gain in the THF-treated mice were significantly lower than those of the control group. THF treatment prevented the increase in body weight and adipose tissue mass. However, increase in glucose and LDL levels in the THF-treated group was observed. These effects may be due partly by the AMPK activators in this extract [12]. 2.2 Anti-Inflammatory and Analgesic Activity Even though a number of steroidal or nonsteroidal anti-inflammatory drugs have been developed, the research of natural compounds as source of new anti-inflammatory agents with lower side effects is a topic of great interest. Nutmeg and mace essential oils are traditionally used to relief sprains and rheumatism. The oil exhibited an anti-inflammatory effect on acute inflammation. An analgesic effect was produced by the oil in the acetic acid-induced writhing model and in the late phase of the formalin-induced licking [13]. A similar effect was observed also with the chloroform extract in a model of inflammation such as carrageenan-induced edema in rats [14]. The anti-inflammatory effects of M. fragrans could be partially due to the presence of myristicin (2) (Fig. 1). This compound did not reduce the cell viability of mouse macrophages (RAW 264.7) but significantly inhibited the production of calcium, interleukin (IL)-6, IL-10, interferon inducible protein-10, monocyte chemotactic protein (MCP)-1, MCP-3, granulocyte-macrophage colony-stimulating factor, macrophage inflammatory protein (MIP)-1α, MIP-1β, and nitric oxide (NO) [15]. Other promising anti-inflammatory agents from nutmeg are macelignans. These compounds suppressed lipopolysaccharide-induced activation of the Toll-like receptor 4 pathway. These actions are mediated by suppression of the nuclear factor NF-κB, reduction in cyclooxygenase type-2 (COX-2) expression, and inhibition of reactive oxygen species generation [16]. Moreover, macelignan (1) inhibited the calcium influx, degranulation, release of histamine, and other inflammatory mediators [17]. The anti-inflammatory activity was exerted also by malabaricone B (3) since it is able to attenuate the increased nitric oxide synthesis and enhancing the arginase pathway in mice [18]. Nutmeg is a traditional remedy for pain. Zhang et al. [19] demonstrated that nutmeg oil administration alleviate the induced joint swelling, mechanical allodynia, and heat hyperanalgesia in rats with a mechanism of action involving reduction of COX-2 expression and blood substance P level. An anti-pain action was demonstrated also for M. fragrans seed kernels alkaloids fraction [20]. At the dose of 1 g/kg this fraction significantly reduced the number of writhing responses in female but not male mice. However, a slightly toxic with LD50 value of 5.1 g/kg was found. 2.3 Antitumor Several research articles evidenced the antitumor activity of M. fragrans with particular reference to its essential oil. Piras et al. [21] isolated volatile and fixed oils from nutmeg. Myristicin (2) (32.8%), sabinene (16.1%), α-pinene (9.8%), and β-pinene (9.4%) were identified as main compounds. In the fixed oil, the most represented fatty acids were myristic acid (14:0), oleic acid (18:1 n-9), and palmitic acid (16:0). Both oils and myristicin (2) displayed a significant growth inhibitory activity against the colon cancer cell line Caco-2. A promising cytotoxic activity was observed with M. fragrans essential oil against human colorectal carcinoma (HCT-116) and human breast carcinoma (MCF-7) cell lines with IC50 values of 78.61 and 66.45 μg/mL, respectively [22]. M. fragrans essential oil showed antiangiogenic activity with IC50 of 77.64 μg/mL maybe due to the presence of some potential antiangiogenic compounds such as myristicin (2), limonene, eugenol, and terpinen-4-ol. Myristicin (2) induced cytotoxicity in human neuroblastoma SK-N-SH with IC50 value > or = 0.5 mM at cells by an apoptotic mechanism since an accumulation of cytochrome-c and the activation of caspase-3 was displayed [23]. The apoptotic pathway with mitochondrial membrane potential alteration, cytochrome c release, caspase-3 activation, PARP-cleavage, and DNA fragmentation was observed, also, when myristicin (2) was applied to human leukemia K562 cell culture [24]. Previously, Chirathaworn et al. [25] observed that M. fragrans methanol extract at concentration of 10 μg/ml induces apoptosis in Jurkat leukemia T cell line through SIRT1 mRNA downregulation. Interestingly, M. fragrans antitumor activity could be mediated, also, by the inhibition of Lactate Dehydrogenase A (LDH-A). Tumor cells predominantly produce ATP by maintaining a high rate of lactate fermentation, rather than by maintaining a comparatively low rate of tricarboxylic acid cycle. M. fragrans seeds extract suppressed cell growth and the overall Warburg effect in human colon cancer cells (HT29). Even though the expression of the enzyme was not changed, both lactate production and LDH-A activity were decreased [26]. Moreover recently, Li et al. [27] demonstrated that nutmeg-treated Apcmin/+ mice had decreased IL-6 levels and normalized dysregulated lipid metabolism, suggesting that uremic toxins are responsible, in part, for the metabolic disorders that occur during colon cancer tumorigenesis. Other M. fragrans essential oil representative compounds such as D-limonene, terpinen-4-ol, and eugenol showed cytotoxic activity through apoptosis-inducing effects on several cancer cells [28]. 2.4 Antimicrobial and Antifungal Activity The use of essential oils from spice is an attractive alternative method to control food/feed microorganisms as they should in principle not be toxic to man and could replace toxic synthetic compounds. The use of nontoxic natural compounds is attributed to growing problems encountered with microbial resistance towards conventional preservation with synthetic compounds and an increasing demand for minimal processed food along with "green" image policies of food industry. M. fragrans is known to exhibit strong antimicrobial activity against food poisoning and spoilage bacteria including Bacillus subtilis, Escherichia coli, Saccharomyces cerevisiae, and multi-drug resistant Salmonella typhi and Helicobacter pylori [29]. More recently, Firouzi et al. [30] reported the effect of nutmeg essential oil on growth and survival of Yersinia enterocolitica and Listeria monocytogenes. Nutmeg oil also had a significant effect on inhibiting the growth of Escherichia coli and Staphyloccocus aureus [31]. Several studies demonstrated also the antibacterial activity of nutmeg extracts. In particular, nutmeg acetone extract has shown the strongest antibacterial and antifungal activity with Staphylococcus aureus (13.8 mm) and Aspergillus niger (14.4 mm), respectively [32]. The nutmeg ethyl acetate extract showed a strong antibacterial activity against gram-positive and gram-negative bacteria with mean MIC value ranging from 0.625 to 1.25 mg/mL, and bactericidal effects at mean MBC value ranging from 0.625 to 20 mg/mL [33]. A good antibacterial activity was obtained also with seed and mace ethanol extracts. M. fragrans seed methanol extract had a strong MIC value of 12.5 μg/ml against the gram-negative bacterium Helicobacter pylori that is recognized as the primary etiological factor associated with the development of gastritis and peptic ulcer disease [34]. A promising activity against H. pylori was found also with dihydroguaiaretic acid (4) (Fig. 2) with MIC of 100 μg/ml and MBC of 125 μg/ml. This effect is comparable with that of clarithromycin [35]. Resorcinols, malabaricone B (3), and malabaricone C (5) from mace also showed strong antibacterial and antifungal activities while macelignan (1) is a potent natural anti-biofilm agent against oral primary colonizers Streptococcus sanguis and Actinomyces viscosus [36, 37]. Nutmeg essential oil showed also antifungal activity against Colletotrichum gloeosporoides, Colletotrichum musae, Fusarium oxysporum, Fusarium semitectum, Aspergillus niger, and Aspergillus glaucus [38]. Previously, Cho et al. [39] evidenced that erythro-austrobailignan-6, meso-dihydroguaiaretic acid and nectandrin-B isolated from the M. fragrans seeds methanol extract exhibited antifungal activities against Alternaria alternata, Colletotrichum coccodes, Colletotrichum gloeosporioides, Magnaporthe grisea, Agrobacterium tumefaciens, Acidovorax konjaci, and Burkholderia glumae. A significant antifungal activity was found also for mace methanol extract against Candida albicans and A. niger [40].[image: ../images/438254_1_En_23_Chapter/438254_1_En_23_Fig2_HTML.png] Fig. 2Dihydroguaiaretic acid (4), malabaricone C (5), licarin A (6) and Machilin A (7) 2.5 Effects on Central Nervous System Several studies demonstrated different effects of M. fragrans in Central Nervous System (CNS). Sonavane et al. [41] investigated the anxiogenic activity of the M. fragrans seed n-hexane extract seeds, acetone-insoluble part of the n-hexane extract, and trimyristin. Both extract and trimyristin reduced the number of head pock in the hole-board test. Authors suggests a nonspecific anxiogenic activity of TM and a mechanism, which involves serotonin (5-HT) and gamma-Aminobutyric acid (GABA). Successively, the same n-hexane extract at the dose of 10 mg/kg dose was found to have comparable potency to imipramine (15 mg/kg) and fluoxetine (20 mg/kg). The antidepressant-like effect of the n-hexane extract involved adrenergic, dopaminergic and serotonergic systems. The n-hexane extract inhibited both α1 and dopaminergic receptor antagonists as well as a serotonin synthesis inhibitor [42]. The memory enhancer activity of M. fragrans is confirmed by the in vitro and in vivo inhibition of acetylcholinesterase [43]. Cuong et al. [44] investigated the cholinesterase inhibitory activity of M. fragrans seeds MeOH extract and n-hexane, ethyl acetate, n-butanol, and water-soluble fractions. EtOAc showed the highest inhibitory potential was submitted to chromatographic purification led to the isolation of thirteen compounds. [(7S)-8′-(4′-hydroxy-3′-methoxyphenyl)-7-Hydroxypropyl]benzene-2,4-diol) exhibited the most effective activity with an IC50 value of 35.1 μM. Values of 42.1 and 44.0 μM were found for [(8R,8′S)-7′-(3′,4′-methylenedioxyphenyl)-8,8′-dimethyl-7-(3,4-dihydroxyphenyl)-butane] and malabaricone C (5), respectively. More recently, Abdul Wahab et al. [45] evidenced that malabaricone B (3) (1.84 and 1.76 μM, respectively) and C (1.94 and 2.80 μM, respectively) are dual and mixed-type inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Molecular docking studies evidenced that both compounds interacts with the peripheral anionic site (PAS), the catalytic triad, and the oxyanion hole of the AChE. Differently Malabaricone B (3) interacted with the PAS, the catalytic triad, and the oxyanion hole of the BChE, while malabaricone C (5) interacted only with the catalytic triad and the oxyanion hole. Anecdotal reports of M. fragrans use as a cheap marijuana substitute, coupled to previous studies reporting a cannabimimetic-like action, suggest that nutmeg may interact with the endocannabinoid system. M. fragrans seeds n-hexane extract administered orally in three doses 5–20 mg/kg p.o. enhanced learning and retention capacities of both young and aged mice. The effect of M. fragrans on endocannabinoid system was confirmed by El-Alfy et al. [46]. Dichloromethane and ethyl acetate fractions prepared from the methanol extract of powdered whole nutmeg interacts with the endocannabinoid system via inhibition of the endocannabinoid catabolizing enzymes fatty acid amide hydrolase and monoacylglycerol lipase. This mechanism provides insight into reported cannabis-like action as well as expands the potential therapeutic utility of nutmeg. Among nutmeg compounds, myristicin (2) demonstrated to possess anxiogenic effect and may antagonize the actions of benzodiazepine at the GABA A receptors level [47]. Moreover, trimyristin, the main triglyceride constituent of nutmeg, showed the depressant effect in mice [48]. On the other hand, Kiyofuji et al. [49] demonstrated that macelignan (1) treatment protected dopaminergic neurons against the interferon (IFN)-c and LP-induced degeneration by activation of the peroxisome proliferator activated receptor c, which in turns activated arginase 1 enzyme expression. For this reason, maceliganan can be used for the treatment of different neurodegenerative disorders. A neuroprotective effect was observed also for licarin A (6) against glutamate-induced toxicity of rat cortical cells [50]. The mechanism of action involves antioxidant effect, reduction of NO, and suppression of Ca2+ influx. In order to clarify the potential central nervous system effects of nutmeg, Wu et al. [51] investigated the permeability of blood-brain barrier of twelve lignans and three phenolic malabaricones from the seeds of M. fragrans. Benzonfuran-type, dibenzylbutane-type, and arylnaphthalene-type lignans showed poor to moderate permeabilities; those of 8-O-4′-neolignan and tetrahydrofuran-lignan have moderate to high permeabilities, while the permeabilities of malabaricones were poor. Erythro-2-(4-allyl-2,6-dimethoxyphenoxy)-1-(3,4-dimethoxyphenyl)-propan-1-ol acetate, verrucosin, and nectandrin B were transported by efflux way, while neolignans dehydrodiisoeugenol, myrislignan, and verrucosin permeabilities was passive diffusion. 2.6 Vascular Activity In the treatment and prevention of cardiovascular disorders and strokes, the inhibition of platelet function plays a critical role. Erythro-(7S,8R)-7-acetoxy-3,4,3′,5′-tetramethoxy-8-O-4′-neolignan (EATN), isolated from M. fragrans, inhibited thrombin- and platelet-activating factor (PAF)-induced platelet aggregation without affecting platelet damage with IC50 values of 3.2 and 3.4 μM, respectively. Moreover, this neolignan attenuated intracellular Ca2+ mobilization in thrombin-activated human platelets through increase cAMP levels [52]. A promising protective effect in vascular occlusive diseases was observed, also, with nectandrin B. This lignin activated AMPK in vascular smooth muscle cells (VSMC) and inhibited VSMC proliferation and neo-intima formation, events that are critical in the development of vascular occlusive diseases [53]. 2.7 Other Activities Nutmeg compounds showed other promising healthy effects. Machilin A (7) (Fig. 2) was able to stimulate, in a dose-dependent manner, osteoblast differentiation through activation of the p38 mitogen activated protein (MAP) kinase pathway [54]. The use of macelignan (1) as photo-aging protective agent is well documented. These compounds reduced the collagen synthesis and simultaneously upregulated the matrix metalloproteinases (MMPs) [55]. Moreover, these compounds act as natural depigmenting agent since at 10 μM inhibited melanosome transfer and dendrite formation in B16F10 melanoma cells [56]. Similarly acts licarin E that regulates the expression of MMP-1 and type-1 procollagen in UVB-irradiated human skin fibroblasts through MAPK and TGFβ signaling [57]. The hepatoprotective effect of nutmeg compounds was reported. Morita et al. [58] have reported that myristicin (2) showed a strong hepatoprotective effect with a mechanism of action that involves the inhibition of tumor necrosis factor-α release from macrophages while of macelignan (1) induced activation of the mitogen activated protein kinase (MAPK) signaling pathway, especially JNK and c-Jun [59]. The hepatoprotective effect demonstrated for nectandrin that protect hepatocytes against oxidative injury through the activation of Nrf2/ARE pathway mediated by ERK phosphorylation and AMPK-dependent inactivation of GSK-3β [60]. 3 Conclusions In traditional medicine, nutmeg has long been used as a remedy for several disease including gastrointestinal problems, cancer, infections, rheumatism, and psychological disorders. Despite its traditional uses, the mechanisms underlying its effects remain unclear and further research studies are undoubtedly needed to properly assess the therapeutic potential of this plant and derived products. A perusal analysis of literature evidenced that myristicin (2), macelignan (1), nectandrin A and B, licarin A–E, and malabaricone B and C were well investigated especially for anxiolytic, anti-cancer, anti-inflammatory, and anti-infective effects. Although there is great interest in M. fragrans extract, oil and isolated constituents, limited preclinical studies on nutmeg not only for size but also for design and focus with no pure derived compounds included was observed. However, the evidence that nutmeg represent an in doubt font of secondary metabolites with significant potential as prototype agents for drug discovery, address the research interest on this plant. Further evaluation of bioactive constituents is warranted especially to determine in vivo pharmacokinetic parameters and to monitored nutmeg constituents toxicity. In fact, it is well documented that excessive doses have a narcotic effect, symptoms of delirium, and epileptic convulsions [61]. Moreover, one of the most promising compounds, myristicin (2) is hepatotoxic when ingested in large amounts [62]. Conflicts of Interest The authors declare no conflicts of interest. References 1. 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Ther Drug Monit 28:568–575. https://doi.org/10.1097/00007691-200608000-00013CrossrefPubMed © Springer Nature Switzerland AG 2019 Jean-Michel Mérillon and Kishan Gopal Ramawat (eds.)Bioactive Molecules in FoodReference Series in Phytochemistryhttps://doi.org/10.1007/978-3-319-78030-6_13 34. Bound Phenolics in Foods Liliana Santos-Zea1 , Javier Villela-Castrejón1 and Janet A. Gutiérrez-Uribe2 (1)Tecnologico de Monterrey, School of Engineering and Science, Centro de Biotecnología-FEMSA, Monterrey, Mexico (2)Tecnologico de Monterrey, School of Engineering and Science, Department of Bioengineering and Science, Campus Puebla, Puebla, Mexico Liliana Santos-Zea Email: email@example.com Javier Villela-Castrejón Email: firstname.lastname@example.com Janet A. Gutiérrez-Uribe (Corresponding author) Email: email@example.com Abstract Bound phenolic compounds are widely distributed among several plants, especially cereals. In most of the cases, covalent bonds are formed with polysaccharides, proteins, or lipids. But additionally, hydrophobic interactions may affect their release from the food matrix. Many studies have reported their bioactivity after their release from foods, in most of the cases involving acid or basic hydrolysis and further extraction with organic solvents. Besides their antioxidant activity, bound phenolics have important effects on the inhibition of cancer cell growth, key enzymes involved in the metabolism of carbohydrates, as well as in the regulation of inflammatory processes. Food processing and gastrointestinal digestion affect the bound phenolic bioavailability and in consequence the potential benefits to human health. Recent studies have demonstrated that microbiota composition in the gastrointestinal tract affect the release of bound phenolics and their metabolism. Therefore future studies will help us to understand the complex interactions between bound phenolics and gastrointestinal microbiota and produce natural controlled released bioactive phenolic compounds. Keywords Bound phenolicsAntioxidantPrebioticExtraction 1 Introduction Phenolic compounds (PCs) are plant secondary metabolites necessary for physiology and cellular metabolism. Among the main functions in plants are the protection against insects and animals, structure, growth, and reproduction [1]. PCs come from the phenylpropanoid pathway, and the two main precursors are the amino acid phenylalanine from the shikimate pathway and malonyl-CoA from the acetate pathway [2]. The phenolic acids possess one or more aromatic rings hydroxyl-substituted. Currently, there are 8000 known phenolic structures, which are classified according to the carbon chain (Fig. 1) [3]. Furthermore, the location within the plant and the chemical structure makes possible to categorize phenolic compounds as soluble and insoluble. Soluble phenolics are composed of free simple phenols, glycosides (with single or multiple sugar moieties), and low molecular weight phenolics not bound to the other plant cell major components. Bound phenolic (BP) group is conformed by condensed tannins, phenolic acids, and other compounds of low molecular weight [4]. Particularly in cereals, the insoluble fraction account for the major part of phenolic acids [5].[image: ../images/438254_1_En_13_Chapter/438254_1_En_13_Fig1_HTML.png] Fig. 1Diversity of phenolic compound structure Phenolic compounds have gained a lot of attention, since these molecules have proved their antioxidant, antimutagenic, antiviral, and antibacterial activities [6–10]. Epidemiological studies have linked the consumption of foods and beverages high in phenolic acids with a reduced risk of development of several diseases [3]. Since bound phenolics are poorly absorbed in the small intestine, their fermentation products generated by the microorganisms in the large intestine generate positive effects on microbiota and inhibit the growth of pathogens. 2 Bound Phenolics in Nature Bound phenolic compounds are widely distributed among several plants, such as cereal grains (>62% of the total phenolic are bound), whereas fruits and vegetables have most of their phytochemicals in the free or soluble form (<24% of the total phenolic are bound) [11]. Phenolics, proteins, and polysaccharides are separated in different compartments in the cell, normally they do not interact until an external stimuli triggers the intracellular contact and then several subsequent reactions can occur and force the interaction (adsorption, oxidation, solubilization, and migration) [12]. PCs are linked to the cell wall structure macromolecules, such as arabinoxylans, pectin, cellulose, lignin, or proteins through ester, ether, and C-C bonds [13]. PCs can interact through hydrogen bonding, covalent bonding, hydrophobic interactions, and ionic bonding with other macromolecules [14]. Some phenolic acids are more abundant in the bound fraction, such as in the case of whole grain oats, where ferulic and p-coumaric acids were found 10- to 100-fold higher in concentration with respect to the free phenolic fraction. However, the profile was less diverse in the bound fraction than in the free phenolics [15]. In the past 20 years, there has been an important increment in research in the field of phenolic interactions with other macromolecules, but there is still limited knowledge about the changes that occur during plant development, postharvest treatments, or interactions produced during food processing and that may affect the release of free phenolics. 2.1 Conjugates with Polysaccharides Phenolic compounds, mainly phenylpropanoids and simple phenolic acids, can be bound to carbohydrates. Hydrogen bonding and hydrophobic interactions are the driving mechanisms for this type of complexes [14]. The non-covalent complexation between cyclodextrin and phenolic acids is one of the most studied interactions. This complexation is driven by the size of cyclodextrin and its polarity [12]. 2.2 Conjugates with Proteins The molecular weight, affinity of the phenolic acid for water, structural flexibility, and the hydroxyl groups are the main characteristics that dictate the binding of phenolic acids with proteins [16]. In addition, this type of reaction is favored under neutral and basic pH conditions. One of the most studied interactions is the complex with the bovine serum albumin (BSA) [17]. Irreversible interactions involve the oxidation of phenolic compounds and the formation of o-quinones or o-semiquinones or the cleavage of proanthocyanidin interflavanic bonds. Hydrolysable tannins are conformed by simple phenolic acids (gallotannins) or hexahydroxydiphenic acids (ellagitannins) esterified to polyols (most commonly glucose). Condensed tannins or proanthocyanidins are polymers and oligomers of flavan-3-ol unit that are distinguished by their degree of polymerization and type of linkage [12]. Tannins can interact with proteins due to hydrophobic interactions between the aromatic ring of the tannin and the hydrophobic region of the protein and the hydrogen bonds. The tannin-protein complexes depend on the size, length, pH, and flexibility of tannins. These molecules are able to precipitate proteins from an aqueous solution. Interaction with PCs can increase the thermal stability of proteins [14, 18, 19]. Protein aggregation is affected by the average size of the condensed tannins rather than the hydroxylation pattern. In special, the procyanidins and cis-flavan-3-ol units contributed most to the tannin interactions on the BSA surface [20]. 3 Bound Phenolic Extraction and Analysis For the precise identification and quantification of phenolic compounds, they need to be correctly extracted. Several extraction processes underestimate the total phenolic compounds due to recovery inefficiency [21]. 3.1 Traditional Methods The most common method is the solid-liquid extraction; it involves the extraction of fresh or freeze-dried materials. But a critical step to extract bound phenolic compounds is their release from the insoluble food matrix. Acid or alkaline hydrolysis has been extensively used for this purpose. Alkaline hydrolysis has been proved to be more effective but time, temperature, and base concentration need to be optimized for each crop. Once the compounds have been released from the matrix and the pH adjusted, extraction with solvents is required to recover bound phenolics. The most used solvents are water, methanol, ethanol, acetone, or several mixes. The parameters in this process are extraction time, temperature, solvent type, solvent to sample ratio, and number of repeated extractions. These parameters must be optimized in order to obtain a substantial recovery of PCs. The main disadvantages of solid-liquid extraction include the need to remove contaminants or other unwanted compounds from the extract, the use of hazardous organic reagents, and the time required for extraction [21]. Traditional food processing releases bound phenolics. Boiling is still the most common cooking method, and it has a negative impact on the total phenolics content of food since bound phenolics are released. After their release, they may form new irreversible covalent bonds with proteins due to the hydrothermal energy [22]. 3.2 Emerging Technologies Several physical methods have been recently used to release bound phenolics from the matrix. They may be used in combination with alkaline hydrolysis. Microwaves, ultrasound, far infrared radiation, pulsed electric field, and pressurized liquids are examples of emerging technologies for the release of bound phenolics [11]. All these methods increase the extraction yield and reduce the hydrolysis time significantly. 4 Bioactive Potential of Bound Phenolics Over the years, diverse biological activities from dietary and medicinal plants have been attributed to phenolic compounds, in both free and bound forms. Antioxidant capacity has been the most widely studied effect of phenolics, but effects, such as antiproliferative and apoptotic effect on cancer cells, inhibition of carbohydrate and lipid metabolism enzymes, and anti-inflammatory and vasodilator effects, among others, have been recognized (Table 1). It is important to point out that in these studies, bound phenolics are initially subjected to hydrolysis, as documented in the previous section, to release the phenolic moiety from the carbohydrate or protein moiety and therefore determine their biological effects.Table 1Potential bioactivity of bound phenolic compounds Source Phenolics Bioactivity Dose/concentration Reference Corn, wheat, oats, rice Ferulic acid and other phenolic acids Chemical antioxidant capacity evaluated by total oxygen scavenging capacity (TOSC) 50–150 μmol vit C eq/g grain [24] Dehulled highland barley Catechin, phenolic acids Cellular antioxidant capacity on HepG2 cells 11.3–19.2 μmol QE/mL [25] Model system of quercetin-BSA Quercetin TEAC antioxidant capacity of BSA-bound quercetin was increased hydrolyzing the protein 2.0–4.6 mM as BSA-quercetin, 3.0–4.0 as peptide-quercetin [26] Rice soluble fiber Ferulic acid bound to arabinoxylans Antioxidant capacity evaluated by inhibition of ß-carotene oxidation (IC50) 1.14–1.24 mg [27] White, red, and tannin sorghum bran, corn fiber Phenolic acids bound to arabinoxylans Chemical antioxidant capacity evaluated by ORAC 20–100 mM Trolox equivalents (TE)/g [28] Cinnamon, oregano, and clove Phenolic acids, cinnamaldehyde, coumarin, eugenol Chemical antioxidant capacity evaluated by FRAP and DPPH methods 2.6–34.6 mmol FeSO4/100 g and 2.3–17.7 mmol TE/100 g [29] Ginger Cinnamic and p-coumaric acids Inhibition of lipid peroxidation (IC50), inhibition of DNA fragmentation 5.2 μg GAE/gliver, 2–4 μg GAE [30] Blackberry, black raspberry, and blueberry meal seeds Phenolic acids, catechins, flavonoids, and anthocyanins Chemical antioxidant capacity, LDL cholesterol oxidation inhibition (48–60% at 22 h), supercoiled DNA strand breakage inhibition (80–98%) 5–68 μmol TE/g, 0.5 mg/mL, 0.10 mg/mL [6] Chlorogenic acid-BSA Model system Binding of chlorogenic acid to BSA inhibits LDL oxidation in a dose-response way 1–10 μM chlorogenic acid +4% BSA [31] Blueberries Phenolic acids, flavonoids, and anthocyanins Cellular antioxidant capacity on HepG2 cells 13.5–63.5 μmol QE/100 g [32] Litchi pulp Phenolic acids, (−)-epicathechin, 4-methylcatechol, rutin, quercetin Cellular antioxidant activity on HepG2 cells, ORAC antioxidant capacity 7.4 ± 1.5 μmol QE/100 g, 286.1 ± 1.5 μmol TE/100 g [33] Cancer treatment or prevention Maize lime-cooking wastewater (nejayote) Feruloyl putrescines, ferulic acid Induction of phase II enzyme quinone reductase 1.28–3.07 μg/mL [34] Dehulled highland barley Catechin, phenolic acids Antiproliferative activity on hepatic cancer HepG2 cells (IC50) 66.4–159.4 mg/mL [25] Cactus cladodes (Opuntia ficus-indica) Isorhamnetin glycosides Apoptotic activity on colon cancer HT-29 (IC50) cells, mediated by caspase 3/7 4.9–34.8 μg/mL [35] Tea catechins encapsulated in milk Epigallocatechin gallate bound to casein micelles Antiproliferative activity on colon cancer HT-29 cells (IC50) 10–20 μg/mL [36] Foxtail millet (Setaria italica) bran Phenolics Apoptotic activity on colon cancer HCT-116 cells, mediated by ROS pathway, and inhibition of NF-κB pathway 0.28–1.43 mg/mL. 0.57–0.88 mg/mL, 0.5–0.7 mg/mL [37] Carbohydrate and lipid metabolism Citrus fruits Phenolic acids, hesperidin Inhibition of α-glucosidase (49.3–80.7%) Not reported [38] Foxtail and little millet p-coumaric, caffeic and ferulic acids, quercetin Inhibition of α-amylase (IC50) and α-glucosidase (IC50) 38.3–54.3 FAE μg/mL, 31.3–35.5 FAE μg/mL [39] Whole grain oats and products Phenolic acids Modulation of maltose hydrolysis (49–61%) 34–85 GAE μg/mL and 8.5–17 GAE μg/mL [15] Inhibition of intestinal glucose transport (31–67%) Shaddock (Citrus maxima) peels Not reported Inhibition of α-amylase (12.9–64-2%) and α-glucosidase (45.9–95.9%) 80–320 μg/mL [40] Soybeans Not reported Inhibition of α-amylase (IC50) and α-glucosidase (IC50) 320.5 and 458.7 μg/mL [41] Black quinoa seeds Phenolic acids and flavonoids Mild inhibition of α-glucosidase (IC50) and pancreatic lipase (IC50) 37.6–55.6 mg/mL and 9.0–10.5 mg/mL [42] Grapefruit peel Resveratrol, caffeic acid, ellagic acid, and other phenolic acids HMG-CoA reductase (IC50) inhibition 115.3 μg/mL [43] Inflammation and cardiovascular health Cactus cladodes (Opuntia ficus-indica) Isorhamnetin glycosides In vivo decrease of cell infiltration (51.8%); inhibition of: NO• (77.2–81.4%), COX-2 (76.3–77.7%), TNF-α (85.2%), and IL-6 (53.0%) 5 mg/kg (rat animal model) [44] Antihypertensive and antiulcerative Grapefruit peel Resveratrol, caffeic acid, ellagic acid, and other phenolic acids Angiotensin converting enzyme (ACE) inhibition (IC50) 137.4 μg/L [43] Raw and cooked pigmented and nonpigmented rice Not identified ACE inhibition (40%) by pigmented rice and (20%) by nonpigmented rice extracts 8 mg FAE/mL. 4 mg FAE/mL [45] Ginger Cinnamic and p-coumaric acids Parietal cell proton pump inhibition (IC50), Helicobacter pylori growth inhibition (MIC) 1.5 μg/mL, 38 μg/mL [30] With respect to in vivo potential, bound phenolics are essentially considered non-bioavailable, as they reach the colon still covalently linked. In general, it has been determined that only about 5–10% of insoluble bound phenolics are absorbed in the small intestine and the majority reaches the colon intact [13]. These compounds can be released by pH conditions in the stomach, processed by ß-glycosidase in the intestinal border brush cells, transported as glycosides by sugar transporters, or enzymatically released by fermentation of the colonic microbiota [11, 13]. Additionally, during and after absorption, phenolics undergo conjugation by intestinal and liver enzymes, such as methylation, sulfation, glucuronidation, increasing hydrophilicity, and therefore excretion [23]. 4.1 Antioxidant Antioxidant capacity of bound phenolics, particularly in the case of grains, has been well studied [24]. In most cases only the in vitro or chemical capacity as antioxidants has been analyzed by diverse methods, such as total oxygen scavenging capacity (TOSC), ferric reducing antioxidant power (FRAP), or oxygen radical antioxidant capacity (ORAC) (Table 1). To determine the impact that phenolic compounds can have in a real biological system, models such as LDL cholesterol oxidation, supercoiled DNA fragmentation, and cellular antioxidant activity assays have become more widely used in recent years [46]. 4.1.1 Antioxidant Capacity by Chemical Methods With respect to chemical antioxidant capacity of bound phenolics, Adom and Liu (2002) determined that in the case of whole grains, such as corn, wheat, and oats, this fraction corresponds to over 60% of the total antioxidant capacity evaluated by the TOSC assay, and this activity was highly correlated to total phenolics and ferulic acid content in the grains [24]. In the case of dehulled highland barley, antioxidant capacity evaluated by ORAC showed that around 50–60% of total antioxidant capacity corresponded to the bound phenolic fraction [25]. The proportion of bound phenolics with respect to total content may vary among different cultivars or varieties of the same species. In different colored bran rice, it was seen that bound phenolic DPPH radical scavenging capacity represented from 7% to 41% of the total antioxidant capacity of rice, while the ORAC activity varied from 26% to 75% of the total capacity coming from the bound fraction [48]. These results indicated that, in the case of whole grains, evaluating the effect of only free phenolics would underestimate the total antioxidant potential of these particular matrices. Feruloyl arabinoxylans form an interesting group of bound phenolic compounds with antioxidant potential. The antioxidant capacity of arabinoxylans obtained from rice was found to be a synergy between the phenolic and carbohydrate moieties. Lower IC50 values were reported (1.14–1.24 mg) with respect to the predicted amount (54.8–56.9 mg), calculated as the equivalent concentration of ferulic acid bound to the fiber [27]. The authors of this work concluded that ferulic acid is also able to exert antioxidant capacity without needing to be released. Similar results were observed for arabinoxylans extracted from sorghum and maize bran, in which antioxidant capacity in the extracts was attributed to ferulic acid and other bound phenolics to this cell wall component [28]. Since arabinoxylans can be used as ingredients in gluten-free bread formulation, antioxidant capacity of the bound phenolics to the carbohydrates can give added value to such food products [49]. With respect to other non-grain sources, the contribution of bound phenolics to antioxidant capacity is generally lower than 50%, depending on the source. In the case of spices such as cinnamon, oregano, and clove, bound phenolics represented between 3% and 17% of the total antioxidant capacity evaluated by the FRAP method and 3–28% by the DPPH method [29]. In the case of litchi pulp, variation in total antioxidant capacity and the proportion represented by bound phenolics was variable among different cultivars, from 10% to 49% of the antioxidant capacity measured by FRAP assay, and EC50 values in DPPH method demonstrated that bound phenolics were three to ten times less effective than free phenolics [50]. Similar results were obtained for litchi pulp when using the ORAC method, where bound antioxidant capacity was reported as 286.1 μmol TE/100 g, while free ORAC values reached up to 3406.8 μmol TE/100 g, representing 7.7% of the total antioxidant capacity [33]. Therefore, in general terms, contribution of bound phenolics to antioxidant capacity of herbs and fruits is lower when compared to cereals and other grains. However, in fruit seeds, a higher amount of antioxidants can be found in the bound fraction. For example, 35–70% of total antioxidant capacity from blackberry, black raspberry, and blueberry seed meals were accounted after hydrolyzing bound phenolics [6]. Binding of PCs to proteins , such as bovine serum albumin (BSA), can affect their antioxidant potential. Such was the case with a quercetin-BSA complex, which had a lower Trolox equivalent antioxidant capacity (TEAC) (2.0–4.6 mM), at concentrations from 7.9 to 17.5 μg of quercetin/mg of protein, with respect to the same amounts of free quercetin (4.4–6.0 mM). Furthermore, a higher antioxidant capacity (3.0–4.0 mM) of the peptide containing 12.2 μg of quercetin/mg of protein obtained after trypsin hydrolysis with respect to the same amount of free quercetin (5.0 mM) [26]. 4.1.2 Antioxidant Capacity in Biological Systems So far, the antioxidant capacity has been discussed in terms of chemical assays; however, the real protective potential of a compound requires evaluation in more complex systems. Antioxidant activity of ginger (Zingiber officinale) extracts was analyzed by two methods to determine its protective effect on biomolecules. Results showed that cinnamic acid and p-coumaric-rich extract inhibited lipid peroxidation (48–60%) at 5.2 μg gallic acid equivalent/g rat liver homogenate (μg GAE/gliver) and protected against DNA oxidative fragmentation (80–98% inhibition) when adding 2–4 GAE μg [30]. Blackberry, black raspberry, and blueberry seed meal extracts at 0.5 mg/mL inhibited LDL cholesterol oxidation from 46% to 60% after 22 h incubation, and DNA strand breakage was inhibited at levels up to 98% with addition of 0.10 mg/mL of extract [6]. Cellular antioxidant activity is a measurement that takes into account the internalization of the compounds into the cell, therefore, making a more realistic approach to the actual antioxidant activity of a given compound or extract in a living system [51]. This assay has been used to evaluate the antioxidant potential of sources, such as blueberry extracts, where CAA values were found in the range of 13.5–63.5 μmol quercetin equivalents (QE) per 100 g fresh weight of berries, from which 8% to 20% of the total phenolic content corresponded to the bound fraction [32]. In the case of litchi pulp, bound phenolic fraction exerted a lower antioxidant capacity (7.4 ± 1.5 μmol QE/100 g) than its corresponding free fraction, which reached up to 56.7 μmol QE/100 g, where bound fraction represented 11% of the CAA [33]. As described with respect to chemical antioxidant evaluation, PCs bound to proteins have also exhibited activity in other models. One example is the inhibition of LDL cholesterol oxidation when incubated in the presence of 4% BSA solutions with different doses of chlorogenic acid. It was shown that up to 80% delay in oxidation initiation occurred when 10 μM chlorogenic acid was used in the system, after removing the non-bound phenolic acid from the solution [31]. This study shows promising activity for these complexes, particularly when considering that protein-binding can confer protection during the digestion process but allowing release and increase of bioavailability, as well as stability during storage [16, 52]. In general, these studies have been conducted after hydrolyzing the bound phenolic fraction from the matrix, by any of the methods discussed in Sects. 3.1 and 3.2. This approach allows us to determine whether bound phenolics should be taken into account when evaluating the antioxidant capacity of a given food source. However, when considering bioavailability, bound phenolics are usually not released and absorbed during gastrointestinal digestion [23]. Food processing such as fermentation, malting, extrusion, or cooking allows the release of this fraction, increasing its uptake and making it available to target organs [11]. Therefore, most of the classical assays underestimate the true antioxidant capacity of foods since most of the compounds responsible of this activity are bound to insoluble matrices. Most of the research on the total antioxidant capacity of foods has ignored the interactions that may occur due to the coexistence of multiple antioxidants. In fact, the multiple extraction procedures used as a preliminary step for classical antioxidant determination hinder the generation of a standardized database. "QUENCHER" (Quick, Easy, New, CHEap, Reproducible) is a simple and direct assay for antioxidant capacity measurement that is extraction-independent and may enable interlaboratory comparison [47]. With this method, the functional groups of the soluble and insoluble bound antioxidants in solid samples make contact with free radicals via the liquid-liquid type of reaction and solid-liquid type of reaction, respectively [47]. 4.2 Cancer Antioxidant mechanisms are proposed as one of the primary ways in which PCs exert their bioactive potential [3]. However, there is evidence of additional anticancer mechanisms for preventive and prophylactic effect of PCs against cancer (Table 1). One of the ways in which BPs can contribute to chemoprevention of cancer is inducing phase II enzymes, such as NADPH-dependent quinone reductase (QR), which work in vivo to inactivate free radicals and electrophiles [53]. Acosta-Estrada et al. (2015) determined that a bound phenolic extract obtained from maize lime-cooking wastewater solids was able to induce QR activity at a concentration of 76 μg/mL and presented a chemopreventive index of 4.58 (cytotoxicity requires a 4.58-fold higher concentration). In this study, QR induction was attributed to coumaroyl-feruloyl and di-feruloyl putrescines that were able to induce QR at 1.28 μg/mL and 3.07 mg/mL, respectively [34]. Antiproliferative effect of BPs released from different sources has been assayed on diverse cell lines. Catechins and phenolic acids from dehulled highland barley showed antiproliferative potential on hepatic cancer HepG2 cells (66.4 μ≤ IC50 ≤ 159.4 mg/mL), observing variation among four barley varieties [25]. In this study, a lower bioactivity occurred with bound phenolics with respect to free phenolics. Glycosylated flavonoids released by alkaline hydrolysis from Opuntia ficus-indica cactus pads and subsequently purified presented a high antiproliferative effect. Alkaline crude extracts were effective on HT-29 (IC50=4.9–9.1 μg/mL) and Caco-2 (IC50=8.2–16.7 μg/mL) colon cancer cell lines. Purified isorhamnetin diglycosides presented IC50 values from 8.6 to 15.2 μg/mL on HT-29 and 25.8–36.7 μg/mL Caco-2 [35]. The authors determined that glycosylation pattern of the isorhamnetin derivatives had an important impact, since diglycosides were more effective than triglycosides [35]. Haratifar et al. (2014) demonstrated that PCs capacity to bind to protein could allow encapsulation of compounds, such as epigallocatechin gallate, in casein micelles, while preserving antiproliferative activity on HT-29 cells (16 ≤ IC50 ≤ 20 μg/mL) [36]. This PC-protein complex could be an alternative for delivery of PCs as a food ingredient or dietary supplement, such as fortified breads, dairy products, and processed meats [16, 52]. Besides the evaluation of the antiproliferative effect, some mechanisms related to the anticancer effect of BPs have been explored. In the case of Opuntia ficus-indica alkaline extracts, an increase in caspase 3/7 was observed by flow cytometry in HT-29 (33%) and Caco-2 (28–43%) cells treated with the extracts when compared to non-treated cells. Moreover, apoptosis was observed in 32–53% of HT-29 cells treated with isorhamnetin diglycosides and 59% when crude extract was used [35]. On the other hand, bound phenols from foxtail millet induced apoptosis by generation of reactive oxygen species (ROS) in HCT-116 colon cancer cells at concentrations from 0.57 to 0.88 mg/mL. Additionally, apoptotic effect was shown by a decrease in Bcl-2 protein and increase in BAX protein, along with inhibition of the NF-κB pathway [37]. Foxtail millet bran contains BPs such as catechin, myricetin, daidzein, luteolin, quercetin, apigenin, naringenin, and kaempferol, likely accountable for the bioactive effect [39]. 4.3 Carbohydrate and Lipid Metabolism Phenolic compounds, such as phenolic acids and flavonoids, are known inhibitors of starch-hydrolyzing enzymes α-amylase and α-glucosidase, capable of delaying carbohydrate digestion [54, 55] or even absorption by glucose transporters [15]. Bound phenolics also have potential to interfere with the activity of the fat-digesting enzymes known as lipases, due to PCs affinity with proteins, diminishing the rate of fat absorption [56]. The BP fraction of several citrus fruits showed α-glucosidase inhibitory activity [38]. The main compounds found in these extracts were ferulic acid, p-coumaric acid, vanillic acid, and the flavonoid hesperidin. It can be noted that the authors reported in the same study that these extracts activated α-amylase and angiotensin converting enzyme (ACE) instead of inhibiting them [38]. Other authors report a strong inhibitory potential, as observed with BP extracts from foxtail (IC50=35.21–35.26 μg ferulic acid equivalents per mL) (μg FAE/mL) and little millet (IC50=38.27–46.9 μg FAE/mL); in both cases the extracts were obtained from hull and bran [39]. Pradeep and Sreerama (2017) determined that p-coumaric, caffeic, and ferulic acids were the major component of the extracts. Additionally, α-glucosidase inhibition was observed for BPs obtained from whole grain oats, with a 49.6–61.3% decrease in maltose hydrolysis at 34–85 μg gallic acid equivalents per gram (GAE μg/g). In this case, ferulic acid represented from 52% to 89.3% of bound phenolics from whole grain oats [15]. The effect of black quinoa seed BPs was weaker, with IC50 in the range between 37.6 and 55.6 mg/mL, and the main components of the extract were phenolic acids and flavonoids [42]. With respect to α-amylase, inhibition by PCs has also been reported. Foxtail and little millet showed IC50 values of 41.7 and 38.3 FAE μg/mL, respectively, in the hull fraction and 54.3 and 46.9 FAE μg/mL in the bran fraction [39]. Bound phenolics from the citrus shaddock (Citrus maxima) fruit peel were able to inhibit α-amylase activity up to 64.2% in a dose-dependent manner, at concentrations from 80 to 320 μg/mL [40]. Soybean BP extracts were found to inhibit 50% of α-amylase activity at 320.5 μg/mL [41]. In general, phenolics are stronger inhibitors of α-glucosidase than of α-amylase, such as in the case of BPs extracted from little millet and shaddock peels, where significant differences were observed [39]. This difference in BPs effect on both enzymes could be important when considering their use as a therapeutic agent for hyperglycemia, since inhibiting α-glucosidase preferentially can prevent side effects from excess inhibition of α-amylase [40, 41]. However, it should be taken into account that in some cases α-amylase was activated by treatment with BPs, as happened with whole grain oat phenolics and citrus fruits [15, 38]. 4.4 Interactions with Microbiota Bound phenolics help to maintain a "healthy" microbiota . For example, cocoa bound flavanols increased the number of Lactobacilli and Bifidobacteria [57]. Many bound phenolics resist enzymatic digestion (oral, gastric, and small and large intestine phases) and exert their antioxidant activity in the large intestine [58]. But more importantly, their release is slow and continuous [59]. The slow release of bound phenolic compounds could be related to the complexity of the matrix and the diversity of microorganisms required to breakdown the chemical interactions [60]. 5 Conclusions For many years, bound phenolics were considered as poor contributors to improve human health due to their strong interactions with the food matrix and low bioavailability. Particularly in cereals, bound phenolics are major contributors to total phenolic content and antioxidant activity. Most of the studies on bound phenolic compounds involve their extraction after acid or base hydrolysis, and therefore a lot about their chemical structure is known. But when gastrointestinal microbiota is taken into consideration as an important element in the digestibility of dietary fiber, the release of bound phenolics is more complex as well as their potential bioactivity. 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Int J Mol Sci 18:2203Crossref © Springer Nature Switzerland AG 2019 Jean-Michel Mérillon and Kishan Gopal Ramawat (eds.)Bioactive Molecules in FoodReference Series in Phytochemistryhttps://doi.org/10.1007/978-3-319-78030-6_52 69. Use of Nanotechnology for Immobilization and Entrapment of Food Applicable Enzymes Milad Fathi1 , Mehri Karim1 , Soroush Rahimi Khoigani1 and Vahid Mosayebi1 (1)Department of Food Science and Technology, College of Agriculture, Isfahan University of Technology, Isfahan, Iran Milad Fathi (Corresponding author) Email: firstname.lastname@example.com Mehri Karim Email: firstname.lastname@example.com Soroush Rahimi Khoigani Email: email@example.com Vahid Mosayebi Email: email@example.com Abstract Enzyme immobilization is defined as artificially restriction of enzyme mobility that leads to change of its structurer, properties, and activity. Since enzymes are very sensitive, their classical immobilization methods are still limited mostly as a result of reduction of enzyme activity. Because of unique and tunable properties of nanomaterials, they have been increasingly applied as carrier for enzyme immobilization. Using nanotechnology some features, such as multifunctionality, addressability, stability, and multi-compartmentalization, could be improved. In this chapter enzyme immobilization using some nanostructure materials such as nanoparticles, magnetic nanoparticles, nanofibers, nanoflowers, nanoliposome, and solgel silica has been reviewed, and their advantages and disadvantages were reviewed. Keywords NanotechnologyEnzymesNanofibersNanoflowersMagnetic nanoparticlesNanoliposomesSolgel 1 Introduction Enzymes are biological catalysts which accelerate chemical reactions by lowering activation energy [1]. Nowadays enzymes have a wide range of applications in food productions such as cheese, bread, yoghurt, and fermented beverages [2]. These biological compounds have excellent properties such as high activity, selectivity, and specificity that may permit to implement the most complex chemical processes [3, 4]. But use of enzymes, purely in food, will be associated with problems such as (1) the need of a lot of enzymes, (2) high costs, (3) increased residual enzyme left in the product, (4) low stability of the enzyme, (5) higher labor costs, (6) incompatible of the continuous process, and (7) low activity [5]. Therefore a new method called immobilization emerged to overcome these limitations which is the powerful tool to improve almost all enzyme properties [3]. During immobilization, enzymes are attached to an inert and insoluble material such as porous glass, polystyrene, agarose beads, and zeolites by physical (hydrophobic and van der Waals), ionic, or covalent binding [4]. The structures of supports have important effect on performance of immobilized enzymes. Thus, the characteristics and reactive groups of material and immobilization conditions are almost considered in development of new supports [3]. Commonly used methods of enzyme immobilization can be classified into (i) physical methods like adsorption, encapsulation, and entrapment and (ii) chemical methods including electrostatic interactions, chelating with a metal, and covalent coupling. Encapsulation and entrapment techniques are commonly used, but the main problem with these methods is the enzyme leakage and slow diffusional mass transfer of substrates and products within the support material. Immobilization using physical adsorption and electrostatic interactions is easy to run, but there are risks regarding non-specific protein binding and loss of enzymes during operation [6]. The main advantage of using physical immobilization methods is their compatibility with mass production. Chemical coupling methods, on the other hand, are more efficient than physical adsorption and entrapment methods with regard to retention of enzyme activity, while they are more complex [7]. Additionally, these methods suffer from the drawback of potential denaturation and deactivation caused by the distortion of the three-dimensional conformation of the protein as a result of multipoint binding [6]. Altogether, the practical applications of the classic immobilization methods are still limited mostly as a result of the reduction of enzyme activity after immobilization [8]. The reason for that is the change of enzyme conformational integrity which in turn leads to the loss of dynamic properties of enzyme [9]. Application of nanotechnology as a new method leads to some improvement in material properties such as multifunctionality, remote addressability, stability, and multi-compartmentalization [10]. Size reduction of enzyme carrier materials to nanoscale can ameliorate the enzyme features, because this particle size provides minimum diffusional limitation, maximum surface area per unit mass, and high enzyme loading [11]. Enhancement of enzyme stability and activity in some nanostructures such as nanoparticles, nanoflower, nanofibers, mesoporous materials, solgel silica, cross-linked enzyme aggregates (CLEAs)/crystals (CLECs), and single-enzyme nanoparticles have recently been reported [12, 13]. Several studies on the enzyme immobilization on nanosized material of polysulfone nanofibers for extracellular lipase from Penicillium notatum [14], PEGylated poly(urea-urethane) nanoparticles for Candida antarctica lipase [15], carrageenan hydrogel beads for b-galactosidase [16] and lactase [17], single-enzyme nanoparticles (SENs) for α-chymotrypsin and trypsin [18], nanogel for phenoxazinone synthase [19], and silica gel for laccase [20] have been reported. 2 Enzyme Immobilization Enzyme immobilization is defined as artificial restriction of enzyme mobility [21]. During immobilization, enzymes are attached to an inert and insoluble material such as porous glass, polystyrene, agarose beads, and zeolites [22]. As shown in Fig. 1, enzymes can be immobilized on the support by different methods, namely, physical adsorption, covalent bonding, entrapment or encapsulation, and cross-linking [4, 23].[image: ../images/438254_1_En_52_Chapter/438254_1_En_52_Fig1_HTML.png] Fig. 1Physical adsorption (a), covalent bonding (b), entrapment or encapsulation (c), cross-linking (d) 2.1 Physical Adsorption During physical adsorption, enzyme is attached to the support by the interaction of reversible or noncovalent bonding (Fig. 1a) which included ionic, hydrogen bonding, hydrophobic, and van der Waals interactions. Ionic and hydrogen bonding are stronger than hydrophobic and van der Waals interactions. Physical adsorption has several advantages such as simplicity in process that is not affected by the enzyme structure and inexpensive process. Because it is composed of reversible bonding, enzyme can be easily leached [21]. 2.2 Covalent Bonding This immobilization method is composed of covalent attachment between amino acid groups such as histidine, thiol, threonine, and glutamic acid and active groups of solid carrier such as indolyl and imidazole [20] (Fig. 1b). Properties of immobilized enzyme is dependent on the type of active groups and their distribution over the solid carrier [21]. Enzyme leaching in this method is low but structure of enzyme can be changed [4]. 2.3 Entrapment and Encapsulation In this method enzymes are physically confined in the limited space (Fig. 1c) by polymeric matrices, cross-linked arrays, hollow fibers, ultrafilter membranes, and liposomes. Entrapment method has advantages, such as its simplicity in process and reduction of chemical changes of enzymes, while low stability of enzymes, being sensitive to environmental changes which can disrupt capsule, and enzyme leaching are its disadvantages [21]. 2.4 Cross-Linking Because selection of support or carrier and designing immobilization process conditions are time-consuming and more than 50% enzyme activity is lost when it is immobilized on the support, the use of cross-linking is increased. Cross-linking is a carrier-free method in which enzymes are chemically cross-linked together without a solid carrier; therefore its volumetric activity is not decreased [4, 21] (Fig. 1d). Direct cross-linking of enzymes resulted in low activity and weak mechanical stability; therefore this method usually is not used [21]. But recently two methods of cross-linking (cross-linked enzyme crystals and cross-linked enzyme aggregates) emerged to overcome these deficiencies [4, 24]. 2.5 Limitations of Enzyme Immobilization It is necessary to mention that enzyme immobilization has practical limitations such as the cost of support which in some cases is more expensive than enzyme, the amount of loaded enzyme on the support being low, and structural changes of enzyme [21]. 2.6 Selection of Support The structure and morphology of supports have a large effect on the performance of immobilized enzymes. Thus, the characteristics of the support, functional groups, and immobilization conditions are important [3]. Different nanostructures such as nanofibers, nanoflowers, nanoparticles, nanoliposome, and solgel were used successfully as support because they have high surface to volume ratio [25]. One of the important supports or carriers for enzymes is nanofibers. Nanofibers are defined as long fibers with fine diameters which range from several micrometer to 10 nm and are fabricated by electrospinning process [26]. 3 Enzyme Immobilization on Nanofibers The term electrospinning is derived from "electrostatic spinning" [27] and is an electrohydrodynamic process which converts a polymeric solution to fibrous material. Electrospinning process is composed of different parts such as power supply, syringe pump, and grounded collector. Polymer solution is ejected from needle syringe with constant rate, and then the polymer is stretched by electrostatic force which exists between the needle (as positive pole) and collector (as negative pole). This phenomenon causes decrease in fiber diameter and solvent evaporation. Figure 2 shows schematic of electrospinning instruments [22, 28].[image: ../images/438254_1_En_52_Chapter/438254_1_En_52_Fig2_HTML.png] Fig. 2Schematic electrospinning process Electrospinning has powerful ability to produce fibers in the range of micrometers to 10 nm. Fibers produced by this method have unique properties such as high surface area, alterable surface properties, and high porosity which directly correlate with solvent vapor pressure. On the other hand, when vapor pressure of solvent is high, the solvent vigorously evaporate from fibers and create many holes or pores on the surface of fibers [23, 29]. Moreover some studies prepared nanofibers with specific secondary structure, which contained core-shell