**5. Bioavailability and Bioaccessibility of Krill Oil Omega-3**

Several studies have investigated the effects of krill oil supplementation on health, and these effects appear to be superior to those of fish oil [88,89]. The superiority of krill oil has been attributed to the higher bioavailability of EPA and DHA, which are in the form of PL [90]. Nevertheless, most of the RCTs conducted to date did not use the same doses of EPA and DHA (for the same outcomes) from krill and fish oil and ignored the differences between the bioactive substances in fish and krill oil [91].

In fact, the minor components contained in krill oil, such as astaxanthin, alphatocopherol, vitamin A and flavonoids, can exert pleiotropic activities and have a positive impact on health, in addition to probably improving the bioaccessibility of EPA and DHA. In this regard, a study by Kohler et al., s reported that EPA and DHA from krill meal had lower bioavailability than krill oil, but the same as fish oil. This study underlines the potential role of minor lipophilic molecules in improving the bioaccessibility, and thus the bioavailability, of EPA and DHA from krill oil [92]. However, the mechanisms of action by which krill oil appears to be superior remain unknown and seem to be closely related to the extract in its entirety and the extraction method.

Long-term RCTs are needed to determine the differences in efficacy and performance of krill oil compared to fish oil. This information is currently incomplete and needs clarification, starting from the role of minor components in the extracts. In addition, EPA and DHA supplemented from fish and krill oils in comparative studies should be at the same dosages and ratio. In fact, in a study by Ramprasath et al., which reported the superiority of krill oil over fish oil (expressed as concentration of n-3 PUFA plasmatic levels), supplementation with EPA and DHA was 777 mg for the krill oil group and 664 mg for the fish oil group [91]. Ulven et al., have demonstrated a change in the EPA/DHA ratio after supplementation with 543 mg of krill oil and 864 mg of fish oil (EPA/DHA ratio: 1.74 for krill group and 1.12 for fish oil group) [12]. Weather this improvement of EPA/DHA ration is an advantage in terms of health effect it is yet to be fully demonstrated.

#### **6. Krill and Metabolic Disorders**

Dietary supplementation with omega-3 fatty acids has been demonstrated to be beneficial for the prevention and/or treatment of cardiovascular diseases (CVD) [93] and possibly other inflammatory and neurological disorders [94,95]. Moreover, increased consumption of EPA and DHA may also be of clinical significance in the prevention and reversal of insulin resistance [96].

The American Heart Association (AHA) recommends a daily intake of at least 1000 mg of omega-3 fatty acids to minimize risk factors associated with CVD, even for patients at high risk of developing CVD. Therefore, despite the great abundance of omega-3 fatty acids available in fish oil, the delivery of fatty acids and presence of astaxanthin in krill oil may provide superior health benefits and meet the AHA recommendations [97].

Krill oil is endowed with a unique chemical composition. Unlike fish oil, it is rich in omega-3 fatty acids present in the form of phospholipids rather than triglycerides. This may be biologically and therapeutically significant, since phospholipids are well-absorbed by the intestine and they are readily incorporated into cell membranes, suggesting that they could be endowed with a more favorable pharmacokinetic profile [98]. Moreover, supplementation with krill oil gives the advantage of not only supplying n-3 PUFAs, but also choline, which is an essential nutrient, since that it is needed in the synthesis of neurotransmitters (acetylcholine) and phospholipids and is important in the transport of lipids and reduction of homocysteine [23]. In addition, krill contains several endogenous antioxidants including astaxanthin (which is responsible for the deep red color), the preservation of krill oil against oxidation and it has potential health-promoting properties [99].

Maki and colleagues observed that a supplementation for four weeks with Antarctic krill oil (2 g/day) increased plasma concentration of EPA and DHA [100]. This suggests it could be used similar to the way fish and fish oil are used, as it is also rich in long-chain omega-3 polyunsaturated fatty acids which contains cardiovascular risk. In this context, supplementation with krill can also represent a promising approach to ameliorate obesity and obesity-associated diseases. To date, preclinical evidence has been collected on the positive impact of krill oil in conditions of metabolic disorders.

It has been also demonstrated that plasma EPA level was significantly greater following krill oil treatment when compared to fish oil treatment. Significant remodeling of the plasma lipidome was observed after four weeks of treatment with krill oil (containing 1.27 g/day of long-chain omega-3 polyunsaturated fatty acids) if compared to fish oil (containing 1.44 g/day of long-chain omega-3 polyunsaturated fatty acids), with a clear differentiation in their effects on different plasma lipids species. In particular the authors reported that more than 38% of the lipids species increased following krill treatment, while only 12% increased when fish oil was used as supplementation [101]. Similar results have been obtained in healthy women over a five hour postprandial period. Clear differences between krill oil and fish oil supplementations in the postprandial period were reported. The most noticeable changes were revealed in diacyl-phospholipids and ether-phospholipids [102].

Finally, subchronic toxicity and genotoxicity studies in rats confirm that krill oil is well-tolerated and seems to be safe with a daily supplementation of 5% [103].

In male Sprague Dawley rats who were fed a high-fat diet (HFD) for two weeks, the consumption of krill oil significantly reduced serum lipids, and the two highest krill oil doses (100 and 200 g/L) also significantly increased HDL levels [104].

In 2009 Cohn's group demonstrated that supplementation of krill oil to HFD mice for eight weeks, dose-dependently reduced hepatic triglycerides, cholesterol and serum, as well as total cholesterol and glucose. Unfortunately, this study was unconclusive regarding the putative mechanism. The peroxisome proliferator-activated receptor (PPAR)α which is supposed to be critical to promote β-oxidation at the liver level, did not significantly change which means it was not involved [105].

The intake of a powder isolated from Antarctic krill showed to ameliorate the hepatic metabolism in a transgenic mouse model of chronic inflammation, for expressing the human tumor necrosis factor-alpha (hTNFα) gene. Lower hepatic and plasma triacylglycerol levels, as well as hepatic gene expression of sterol regulatory element binding transcription factor 2 (SREBP2) and enzymes involved in cholesterol synthesis were found. In addition, genes involved in lipogenesis and glycerolipid synthesis were down-regulated and β-oxidation

was promoted, confirming the capability of this product to increase the hepatic lipid catabolism and suppress lipidogenesis. Finally, krill powder reduced endogenous TNFα in the liver, indicating anti-inflammatory effects [106].

In 2020, Saito et al. evaluated the effects of 8-HEPE-concentrated material from Pacific krill on dyslipidemia and hepatic steatosis in low-density lipoprotein (LDL) receptordeficient (LDLR-KO) mice. Very interestingly, they observed that over 18 weeks a supplementation of a typical western diet with Pacific krill (8-HEPE (100 mg/kg), but not EPA or DHA) improved the lipidic profile (reducing LDL and total cholesterol levels and increasing HDL levels) and reduced hepatic triglyceride levels [107]. This led to the hypothesis that eicosapentaenoic acid (EPA) and 8-hydroxyeicosapentaenoic acid (8 HEPE) have more positive effects on the metabolic syndrome by activating the peroxisome proliferator activated receptor (PPAR α) in the liver.

In addition, in dyslipidemic and diabetic non-human primates, the daily intake omega-3 phospholipids purified from krill showed a positive impact on CVD risk factors by reducing total cholesterol, LDL-cholesterol and triglycerides and increasing HDL-cholesterol, if used at the dose equal to 150 mg/Kg/day [108].

Runbland and colleagues reported a randomized control study carried out on 36 individuals who were divided in three groups: fish group, krill group and control group. For eight weeks, krill and control groups received capsules containing oil, while the fish group was invited to consume lean and fatty fishes based on dietary guidelines. As expected, the levels of EPA and DHA increased in the fish group and krill group, whereas docosapentaenoic acid (DPA) increased only in the krill group. However, the overall differences between the three intervention groups were significant for EPA (*p* < 0.0001), DPA (*p* < 0.001) and DHA (*p* < 0.001). In general, the authors observed a great variability among the participants, however a tendency towards a decrease in total lipids and triacylglycerols was observed in the fish and krill groups with the largest VLDL levels. In agreement with previous works in animal models, in which a down-regulation of the genes involved in gluconeogenesis has been demonstrated [109,110], as well as in clinical studies [111], they reported a significant reduction in fasting blood glucose in the krill group compared with the control group. This result is strongly predictive of a reduction of CVD risk [112].

In humans with borderline or high triglyceride levels (range between 150–499 mg/dL), the treatment with krill oil at the dose of 0.5, 1, 2, or 4 g/day for 6 and 12 weeks could be efficient to reduce triglycerides. Nevertheless, the great heterogeneity of the selected sample impeded to have a clear view of the real nutritional value [113].

Cicero et al., published a randomized cross-over clinical trial in which 25 moderately hypertriglyceridemic subjects (150–500 mg/dL) were treated with omega 3 ethyl ester (2000 mg/day) or krill oil (1000 mg/day) for four weeks. Only the krill oil treatment significantly improved HDL and apolipoprotein AI levels, compared to the values measured both at baseline (*p* < 0.05) and at end of treatment in the group supplemented with esterified omega 3 (*p* < 0.05). Both treatments were able to significantly reduce high-sensitivity C-reactive protein (hs-CRP) levels from the baseline (*p* < 0.05), but krill oil improved it more efficaciously than esterified omega 3 ethyl ester group (*p* < 0.05) [114].

A reduction in body weight because of krill oil supplementation in obesity models has been reported in some animal studies. Sun and colleagues observed that an Antarctic krill oil, extracted from dry krill using an innovative procedure of hot pump dehydration combined with freezing-drying, was endowed with anti-obesity effects in metabolic disorder conditions. In particular, supplementation for 12 weeks improved dyslipidemia, fatty liver and glucose metabolism in C57BL/6J mice fed with HFD. Krill oil also reduced body weight gain, reduced fat accumulation in adipose and liver tissue, lowered serum density of lipoprotein-cholesterol (LDL-C) content and ameliorated glucose tolerance. In addition, krill oil feeding also reduced oxidative damage in the liver [115].

In another study, rats were fed a control diet, an HFD or an HFD supplemented with 2.5% krill oil for 12 weeks. Krill oil significantly prevented increased body weight in the HFD group [105].

An improvement of insulin sensitivity and secretion after administration of krill oil (600 mg/day) has also been seen in an obesity model of castrated male New Zealand white rabbits [110]. Expression levels of key enzymes involved in the β-oxidation and lipogenesis were different after krill oil feeding for 8 weeks, compared to placebo, which ultimately led to decreased fasting blood glucose and improved glucose tolerance in the rabbits.

Further evidence suggested that omega-3 phospholipids of krill oil enhanced intestinal fatty acid oxidation and could contribute to the anti-steatotic effects in obese mice, meaning there could exist an axis microbiota-intestine-liver [116]. Indeed, omega-3 alleviated hepatic steatosis in various rodent models of obesity even in exacerbated hepatic steatosis conditions, in which an HFD was combined with thermoneutral animal housing (i.e., ambient temperature approximately 30 ◦C) [117].

Several authors highlighted that omega-3 supplemented by krill oil is even more effective, mainly phosphatidylcholine -rich phospholipids when compared to the same dose of the triacylglycerol form. It has been observed that omega-3 phospholipids contribute more effectively to improve glucose intolerance and insulin resistance in dietary obese mice when compared to their triacylglycerol form [116]. The reason for this is likely due to the amelioration of bioavailability; EPA and DHA present in fish and fish oil are almost exclusively in triacylglycerol form, while in krill oil up to 65% of EPA and DHA occur in phospholipids [118].

Krill oil was also found to directly influence cardiac remodeling and function in an experimental myocardial infarction (MI). In such experimental conditions, rats were randomized in krill oil or control groups for 14 days before the induction of MI. Seven weeks after the MI induction, the echocardiography showed a significant attenuation of left ventricular (LV) dilation in the group pre-treated with krill oil. Attenuated heart and lung hypertrophy and reduced mRNA levels encoded classical markers of LV stress, including matrix remodeling and inflammation [119].

Krill oil has been also associated with moderate improvement in endothelial dysfunction and HDL, two known CVD risk factors, in patients with type 2 diabetes. In 34 participants with type 2 diabetes, an improvement of their endothelial function and a reduction in blood C peptide levels and HOMA scores were reported after four weeks of supplementation with krill oil (1 g/day in PUFA) when compared to the olive oil group. There were differences in weight loss between krill oil and olive oil after 17 weeks, though if compared with their respective baseline measurements, the participants of each group had a statistically significant improvement in endothelium [120].

A close relationship between krill oil consumption and reduction of circulating levels of endocannabinoids 2-arachidonoylglycerol (2-AG) and N-arachidonoyl-ethanolamine (AEA) has been highlighted [116].

In obese subjects, the endocannabinoids are elevated in the blood and this phenomenon appears to be due to changes in expression of adipose tissue metabolizing enzymes. Moreover, endocannabinoids are made by enzymatic reactions from arachidonic acid; hence, the more omega-6 arachidonic acid (ARA) available, the more endocannabinoids can be made. On the other hand, an increased intake of omega-3 might help to counterbalance a disturbed omega-3 to omega-6 ratio and result in lower endocannabinoid levels that may positively affect membrane signaling and energy metabolism. Recently, Di Marzo and Silvestri brought to light the existence of a triangle among the lifestyle–gut microbiome–endocannabinoid system and its crucial role in the development of metabolic syndrome [121].

Interestingly, krill oil supplementation (dose ranging 1.25–5%) led to a significant decrease in AEA, as compared to controls [122]. According to previous preclinical evidence, Berge and colleagues demonstrated that besides a reduction of plasma triglycerides, supplementation with krill powder (4 g/day per os) contributed to a reduction in 11 obese men's anthropometric parameters and blood endocannabinoid (AEA and 2-AG), whose levels were correlated with high levels of triglycerides and were responsible for hyperactivity of the cannabinoid system which feeds metabolic dysfunction. Indeed, the endocannabinoid

system is deeply involved in the regulation of the homeostasis of body composition by regulating food intake and energy expenditure; therefore, this could be another mechanism through which krill may have beneficial effects on metabolism [123]. According to a previous paper, a significant reduction of the 2-AG levels has been highlighted with 2 g/day of krill oil (providing 309 mg/day of EPA/DHA 2:1), although no significant effect on anthropometric parameters has been observed [124].

### **7. Krill and Inflammatory Bowel Diseases and Gut Microbiota**

It is well known that obesity and its complications, such as insulin resistance, hyperlipidemia and atherosclerosis caused by HFD are often accompanied with alteration in gut microbiota, in particular an increase of pro-inflammatory/pathogenic bacteria. In 2017, Cui et al. observed that treatment with fish oil (600 μg/g/day), krill oil (600 μg/g/day) and their mixture (300 + 300 μg/g/day) for 12 weeks led to obesity alleviation, as well as gut microbiota modulation. In fact, they reported a decrease of body weight gain, adiposity index and liver index. They also reported an increase in the abundance of some positive phyla in the gut, including *Bacteroides* and *Lactobacilli* [125]. Likewise, Lu and colleagues demonstrated that supplementation with krill oil shifted the gut microbiota composition and that it was associated with the alleviation of hyperlipidemia. According to an experimental model, mice fed for 12 weeks with a high fat and high sugar diet showed obesity and hyperlipidemia. Treatment with a high dose (600 μg/g/day) of krill oil, but not with lower doses (100 μg/g/day or 200 μg/g/day) improved the microbiotic alteration and cardiometabolic parameters [126].

Krill oil treatments for seven weeks at different dosages (100, 200 and 600 mg/kg) decreased the abundance of tyrosine consumers and increased the abundance of Lactobacillus spp. and short-chain fatty acids producers [127].

Moreover, other research points out beneficial effects of krill supplementation against inflammatory bowel diseases (IBD), including ulcerative colitis and Crohn's disease, which share common symptoms such as bleeding, diarrhea and weight loss. In these cases, the integrity of the intestinal barrier layer and the gut microbiota play a critical role. In this regard, a mixture composed of krill oil plus probiotic *Lactobacillus reuteri* plus vitamin D has demonstrated to significantly improve clinical and histological scores, restore epithelial restitution and reduce proinflammatory cytokines in an experimental model of colitis induced by dextran sulphate sodium (DSS) treatment [128]. Of note, krill oil also appears to attenuate inflammation in an experimental model of ulcerative colitis in rats. In male rats submitted to treatment with dextran sulphate sodium (DSS), the supplementation with krill oil (5%) for 30 days preserved the colon length, which was significantly shortened in the DSS-treated compared to control animals, in line with oedema and inflammation in the colonial mucosa. Moreover, typical factors such as disease activity index (DAI) and TNF-α and IL-1β levels were positively affected by krill oil compared to DSS administration alone [129].

Suppression of the pro-inflammatory cytokines TNF and IL6 and the systemic levels of endotoxin, a marker of IBD, were also found. Recently innovative krill oil-entrapped liposomes were developed and their efficacy in an IBD model was demonstrated. The authors observed that liposomes were incorporated into the impaired enterocyte membrane, which contributed to re-establish the hydrophobic protective barrier in the inflamed/impaired region and decrease the permeation typical of IBD [130].

Liu and colleagues suggested that krill oil could contribute both to attenuating the inflammatory pathway and modulating gut microbiota through the reduction of Rickettsiales and several species of *Lactobacillus* [131].
