*2.7. Minerals*

Other than the organic compounds responsible for the pharmacological activity of krill oil, there is also a large quantity of minerals that have been characterized in some krill samples. Indeed, one of the most abundant minerals contained in krill is calcium (1322 mg/100 g) which can be exploited for bone health, phosphorus (1140 mg/100 g), and magnesium (360 mg/100 g) [30]. Other minerals are contained in minor quantities such as zinc, selenium and potassium. Besides these essential elements, krill is characterized by a large quantity of fluoride (2400 mg/kg dry matter) which may cause skeletal fluorosis if its intake is high. However, the major quantity of fluoride is contained in the krill exoskeleton which could be removed during krill oil extraction to avoid excessive fluoride content reaching low fluoride levels (<0.5 ppm) [47].

#### **3. Mechanism of Action**

Due to the complex composition of krill oil, which contains structurally different chemical compounds such as PUFAs, flavonoids, astaxanthin and vitamins, the pharmacological effects that have been described are ascribable to multiple mechanisms of action. Krill oil is characterized by a high quantity of (n-3) PUFA (mainly EPA and DHA) which are natural PPAR's ligands responsible for the activation of PPAR [33]. These transcription factors play a fundamental role in regulating cell and tissue behavior to different stimuli. Generally, PPAR forms a heterodimer with the retinoic-X-receptor whose ligand is represented by cis-9-retinoic acid [48]. PPARα and PPARγ are the most investigated isoforms of PPAR. PPARα is mainly expressed in hepatic cells and regulates lipid accumulation [49], while PPARγ has been mainly described in adipose tissues where it promotes insulin sensitivity, adipocyte differentiation and regulates metabolic responses, fat storage and energy homeostasis [50]. Furthermore, PPARγ has also been described in inflammatory cells, where it controls the release of proinflammatory mediators and promotes

anti-inflammatory effects [51]. The PPARγ activation occurs in the cells and the uptake of EPA and DHA seems to be due to the expression of FAT/CD36 (a transmembrane fatty acid transporter) [52]. Intriguingly, PPARγ also regulates the expression of FAT/CD36 itself, indicating that n3-PUFA can increase their own uptake inside the adipocytes, promoting the production of adiponectin [53]. The involvement of PPARγ have been demonstrated using PPARγ antagonists (e.g., bisphenol-A-diglycidyl ether or GW9662) that suppressed the secretion of adiponectin [54]. Furthermore, n-3 PUFA are able to activate PPAR through a non-covalent interaction, promoting the reduction of inflammatory responses with a consequent reduction of the release of TNFα and IL-6 after LPS stimulation [55].

Another important target mediating the pharmacological effects of krill oil is represented by G protein-coupled transmembrane receptors (GPCRs) which are involved in the regulation of many metabolic processes. In particular, EPA and DHA activate GPR120 (also known as FFA receptor 4; FFAR4) leading to the increase of intracellular cAMP level and Ca2+ concentrations, consequently promoting the phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2) [56]. Since EPA and DHA are involved in the regulation of inflammatory processes mainly in adipose tissue, the involvement of GPR120 has been investigated. In particular, DHA inhibited the IKK (Inhibitor of κB kinase) complex activation and JNK phosphorylation resulting in a reduction of TNF-α release in macrophages treated with LPS [57]. The involvement of GPR120 has been confirmed by GPR120 knockdown. In addition, DHA has been reported to facilitate the formation of GPR120 and β-arrestin2 complex (GPR120-βarr2) that blocks the pro-inflammatory stimulus due to LPS exposure [58]. The GPR120-mediated anti-inflammatory effects of DHA and EPA have also been confirmed in 3T3-L1 adipocytes, resulting in a significant reduction in MCP-1, IL-1β and TNF-α gene expression [58].

The inflammatory process is mainly regulated by NF-κB. Once this transcription factor is activated after IκB phosphorylation due to external stimuli such as UV radiation, endotoxins, oxidative stress, saturated fatty acids, it is able to translocate into the nucleus. It can then promote the production of several pro-inflammatory mediators, adhesion molecules, COX-2, and inducible NO synthase [59]. EPA and DHA, as reported above, reduce the production of a variety of pro inflammatory molecules, such as TNFα, IL-1, IL-6, IL-8, and IL-12 and limit the transcription of those enzymes involved in the inflammatory process including inducible NO synthase and COX-2 in different cell lines (for example endothelial cells, macrophages and monocytes) [60]. This effect seems to involve the reduction of IκB phosphorylation and consequently the reduction of the activation of NF-κB in a GPR120 and PPARγ dependent manner. Indeed, PPAR physically interacts with NF-κB, avoiding its translocation into the nucleus. Furthermore, NF-κB activity is also related to GPR120 since DHA strongly inhibited IKK activity via GPR120 in both stimulated macrophages and adipocytes [61].

In addition, treatment with n3-PUFA limited NF-κB DNA binding activity in adipocytes, macrophages and THP-1 monocytes stimulated with LPS, which prevents the production of IL-6, IL-1β and TNF-α [62]. Intriguingly, as along with NF-κB DNA binding activity being reduced, the authors demonstrated that PPARγ DNA binding activity was also significantly abolished, providing evidence of the tight connection between PPARγ and NF-κB activity in regulating the inflammatory processes. Alongside the reduction of pro inflammatory mediators, treatment with EPA and DHA promoted the release of IL10 in 3T3-L1 adipocytes [63]. IL10 is an important interleukin that is involved in the anti-inflammatory response and inhibits IKK, preventing NF-κB DNA binding activity and PPARγ binding motif. This demonstrates that n3-PUFA may regulate NF-κB activity by inducing IL-10 expression in a PPARγ dependent manner [64].

Besides the high content of EPA and DHA, krill oil also contains potent antioxidant molecules. In particular, many studies have demonstrated that the presence of astaxanthin is responsible for the potent antioxidant effect and potentially the well-known anti-inflammatory properties of EPA and DHA. Oxidative stress is the leading cause of many pathological conditions by triggering the activation of important proinflammatory

intracellular pathways which feed a vicious circle. This is especially true in neurodegenerative processes and in cardiovascular diseases characterized by endothelial dysfunction. Preventing the excessive production of reactive oxygen species (ROS) through a nutraceutical approach may be a promising strategy to manage several pathological conditions. In this context, Nrf-2 is one of the main transcriptional factors controlling antioxidant machinery. Indeed, its activation is reported to exert beneficial effects through the increased production of direct antioxidant molecules as well as the hyper activation of antioxidant enzymes SOD, CAT, and GPX [65]. Nrf-2 is one of the main targets of the antioxidant effect of astaxanthin, which induces Nrf2–ARE-mediated antioxidant enzymes in different in vitro models. Astaxanthin reduced oxidative stress in neuronal cells exposed to doxorubicin results in an increase in cell viability and reduction of pro inflammatory mediators [66]. Similarly, astaxanthin protected human mesangial cells exposed to high glucose exert an anti-inflammatory and anti-oxidant effect [67].

Oxidative stress, beyond promoting the inflammatory response, is also responsible for insulin resistance due to the activation of several kinases, among which JNK promotes the phosphorylation of IRS-1, inhibiting its activity and preventing its interaction with the insulin receptor. In addition, elevated levels of ROS cause the degradation of the GLUT4 vesicle, dramatically decreasing glucose uptake [68]. The potent antioxidant effect of astaxanthin facilitates insulin secretion, accelerates glucose metabolism and improves insulin sensitivity, IRS-1 activation, Akt phosphorylation, and GLUT4 translocation in skeletal muscle. This leads to increased insulin sensitivity and a decrease in blood glucose level, which in turn paves the way for using the astaxanthin nutraceutical supply for the management of type 2 diabetes [69] (Table 3).

#### **4. Krill Oil Extraction Technologies**

Krill oil can be extracted from different biomasses of fresh krill and dried krill [70,71]. However, fresh krill contains high levels of proteolytic enzymes that induce the rapid autolysis of the crustacean. For this reason, it is necessary to process krill immediately after it is caught [72]. The extraction techniques (Figure 1) can be divided into classic and non-conventional methods. Conventional techniques include several solvent extractions, while the innovative methods include non-solvent extractions, super- and sub-critical fluid extractions and enzyme-assisted extractions.

**Figure 1.** The advantages and disadvantages of krill oil extraction technologies.
