How does Antarctic krill oil improve astaxanthin bioavailability?

 The Antarctic krill (Euphausia superba) is widely distributed in Antarctic waters, with a biomass of 6.5 to 1 billion t [1], a huge amount of resources, and a huge potential to create China's second offshore fishery. In recent years, offshore resources are facing depletion, the situation is grim, and actively develop pelagic biological resources has become an important strategic choice for the development of China's marine fisheries. Antarctic krill is the largest single species of biological resources in the Southern Ocean, accelerate its development and utilization is of great significance.

 

China's Antarctic krill processing industry started late, the core competitiveness of key technologies is weak, the variety of high value-added products is single, and the driving force for industrial development is insufficient [2]. Accelerating the development of a series of high-value products is the key to promoting the development of the Antarctic krill industry.

 

Antarctic krill oil is Antarctic krill extraction, concentration, filtration and other processes made of oil products, rich in EPA/DHA phospholipids, astaxanthin and other active ingredients [3], compared with traditional fish oil (EPA/DHA triglyceride), it has better physiological efficacy in the regulation of lipid metabolism/glucose metabolism [4-6], inhibition of inflammatory responses [6], improve nerve cell function [7-8] and so on. Internationally, based on Antarctic krill oil, combined with conjugated linoleic acid, coenzyme Q10, vitamin D, probiotic Lactobacillus reuteri and other functional food raw materials [9-11], the development of derivative functional products to position the needs of different groups of people has become the main direction of commercial development of Antarctic krill. Antarctic krill oil and its series of derivative products market value reached hundreds of millions of dollars, directly driving the rapid development of the international krill industry. Finding suitable functional food raw materials, innovative Antarctic krill oil derivatives product development, improve the international competitiveness of the product has become an important direction for the development of China's Antarctic krill processing industry.

 

Astaxanthin, as a non-vitamin A prolipid-soluble carotenoid, is widely distributed in marine food materials such as shrimps, crabs, and microalgae, and is the strongest antioxidant found in nature[12] . Natural astaxanthin exists in the free or esterified state [13-15], with a variety of physiological activities to improve the level of oxidative stress in the body, prevention and treatment of cardiovascular and cerebral vascular diseases and neurodegenerative diseases [15-16], and has attracted great attention in the field of food, functional food, and biomedical products in recent years. Astaxanthin cannot be synthesized in the body and can only be consumed through dietary intake, however, due to its hydrophobic properties, its digestive and absorption utilization in organisms is low [15, 17]. Currently, some studies have pointed out that the addition of lipids is an effective way to improve the bioavailability of carotenoids [18-20], which provides an idea for the development of functional products by combining natural astaxanthin extracts with Antarctic krill oil. In summary, this study intends to establish the quantitative detection of astaxanthin in biological samples on the basis of in vivo digestion and absorption experiments to evaluate the impact of Antarctic krill oil on the bioavailability of natural astaxanthin, aimed at confirming the feasibility of Antarctic krill oil - astaxanthin functional combination of product development for the development of new types of Antarctic krill oil derivatives to provide a theoretical basis.

 

1 Materials and Methods

1.1 Materials and reagents

SPF grade male Wistar rats [Production Permit No. SCXK (LU) 20140007], body weight 180~200 g, Qingdao Daren Fucheng Animal Husbandry Ltd; Astaxanthin extract from Haematococcus pluvialis [Astaxanthin content (16.0 ± 0.0%)], body weight 1.0~2.0 g, Qingdao Daren Fucheng Animal Husbandry Co. (16.0 ± 0 . (16.0 ± 0.57)%], Yunnan Aerogen Biotechnology Co., Ltd; Antarctic krill oil [phospholipid content (49.8 ± 0.52)g/100g, astaxanthin content (188.3 ± 4.71)mg/kg, fatty acid composition of EPA (21.7 ± 0.33)%, DHA (12.5 ± 0.19)%], Qingdao Antarctic Weikang Bio-Technology Co; Triglyceride fish oil [EPA (21.9±0.56)%, DHA (12.6±0.22)%], Jiangsu Aoqi Marine Biological Engineering Co.

All-trans astaxanthin standard (purity ≥95.8 %), Dr. Ehrenstorfer, Germany; methanol, methyl tert-butyl ether and other chromatographic reagents, Merck, Germany; porcine bile salts, Beijing Solebao Technology Co. Ltd.

 

1.2 Instruments and equipment

LC-16 liquid chromatograph (with Essentia SPD-16 UV detector), Shimadzu, Japan; YMC-Carotenoid column (250 mm × 4.6 mm, 5 μm), YMC, Japan; T10 high-speed disperser, IKA, Germany; Neofuge 15R high-speed refrigerated centrifuge, Shanghai Lixin Scientific Instruments Co. Ltd.; CTFD-10P Vacuum Freeze Dryer, Qingdao Yonghe Chuangxin Electronic Technology Co.

 

1.3 Experimental Methods

1.3.1 Formulation of gavage emulsions    

The gavage emulsion was prepared according to the method of literature [21] and according to the experimental group. Appropriate amounts of astaxanthin extract from red algae, Antarctic krill oil and fish oil were weighed and added into 0.5% porcine bile salt solution, and then ultrasonicated for 5 min, and homogenized for 5 min at 10 000 r/min using a high-speed dispersion homogenizer to form a homogeneously dispersed emulsion, i.e., the gastric lavage emulsion.

 

1.3.2 Animal grouping and feeding     

SPF male Wistar rats, after 1 week of adaptive feeding, were randomly divided into four groups: blank control group, astaxanthin group, Antarctic krill oil + astaxanthin group, fish oil + astaxanthin group, 50 rats in each group. According to the daily dietary intake of adults (Antarctic krill oil 2 g/d or fish oil 2 g/d, astaxanthin 12 mg/d) was converted to rat dose, the corresponding fat emulsion was given by gavage, and the blank control group was given an equal amount of pig bile salt solution as a control. 实验前 12 h 禁  食 、不 禁 水 ，于 灌 胃 后 0，0.5，1，2，4，8，10，12，16，

After 24 hours of anesthesia with 3% sodium pentobarbital, blood was collected from the abdominal aorta and then killed. The serum was separated, and the intestinal tract (from the pylorus of the stomach to the tip of the cecum) was peeled off and the intestinal contents were collected by saline rinsing, and the small intestine was partially dissected longitudinally, and the small intestinal villi were scraped out. Then the small intestine was dissected longitudinally and the villi were scraped. The biological samples, including serum, villi and intestinal contents, were frozen in liquid nitrogen and placed in a -80 ℃ freezer for spare parts.

 

1.3.3 Determination of astaxanthin content

1.3.3.1 Sample pre-treatment  

  The extraction of astaxanthin from biological samples was carried out by the methods described in [22] to [24], avoiding light as much as possible.

Pre-treatment of serum samples: take appropriate amount of serum, add 5 times the volume of trichloromethane-methanol solution (2:1, v/v), vortex extraction for 1 min, let it stand for 5 min, centrifuged at 8000 r/min for 5 min at 4 ℃, collect the lower layer of the solvent, repeat extraction for 3 times, and then combine the extraction solution, filtered by 0.45 μm membrane and blown dry by nitrogen, then re-dissolve the sample by using methanol-methyl tert-butyl ether solution (1:1, v/v) and leave it for testing. The solution was re-dissolved in methanol-methyl tert-butyl ether solution (1:1, v/v) and left to be measured.

After vacuum freeze-drying, the small intestine villi were ground and crushed, then added with trichloromethane-methanol solution (2∶1, v/v) and extracted by vortex for 1 min, then allowed to stand for 5 min, then centrifuged at 8000 r/min for 5 min at 4 ℃, and the supernatant was collected and the extraction was repeated three times. The extracts were then dried under nitrogen, re-dissolved in methanol-methyl tert-butyl ether solution (1∶1, v/v), and left to be tested.

Pre-treatment of intestinal contents: After vacuum freeze-drying, the intestinal contents were ground and crushed, and then added with trichloromethane-methanol solution (2∶1, v/v/v) and vortex extracted for 1 min, and then centrifuged at 8000 r/min in 4 ℃ for 5 min after 5 min of resting time. The supernatant was collected, and then the extraction was repeated for three times, and then the extracts were combined and fixed to be 25 mL. 2 mL of the extract was transferred into a 10 mL volumetric flask, and 0.5 mL of 0.1 mol/L sodium hydroxide-methanol solution was added, sealed with nitrogen, and saponified at 4 ℃ for 12 h. After neutralization of the remaining alkaline solution with 2% phosphoric acid-methanol solution, the mixture was mixed well, and then fixed at 10 mL, and filtered by 0.45 μm filter membrane for measurement.

 

1.3.3.2 Chromatographic conditions[24]      

Column: YMC-Carotenoid column (250 mm × 4.6 mm, 5 μm); mobile phase A: methanol; mobile phase B: methyl tert-butyl ether; mobile phase C: 1% phosphoric acid solution; gradient elution program in Table 1. Flow rate: 1.0 mL/min; injection volume: 20 μL; detection wavelength: 474 nm; quantification by external standard method.

 

1.4 Calculation of bioavailability

The area under the curve (AUC0-t) was calculated by the trapezoidal area method according to the curve of serum astaxanthin content, and the size of AUC0-t was used to measure the bioavailability of astaxanthin in different experimental groups[25-26] .

 

1.5 Data processing

The experimental data were "mean ± standard deviation", and were processed and statistically analyzed using Excel 2016, SPSS Statistic 20 and other software. One-way ANOVA (Tukey) was used for two-way comparisons between different experimental groups, and statistically significant differences were defined as P<0.05.

 

2 Results and analysis

2.1 Methodology for the quantitative determination of astaxanthin in biological samples

2.1.1 Limit of detection and linear range    

The limit of detection (LOD) was 0.05 μg/mL with a signal-to-noise ratio (S/N) of 3. In the mass concentration range of 0.10~5.0 μg/mL, the linear relationship between astaxanthin concentration (X) and the peak area (Y) of the instrumental response was good, with Y=240735X-7526.3 (R2=0.9993), which can be accurately quantified by the external standard method.

 

2.1.2 Recovery and Precision    

The separation of astaxanthin in different biological samples is shown in Figure 1. Under the determined conditions, the peak time of astaxanthin standard was 10.122 min, and astaxanthin was well separated from other impurities in rat serum, small intestine villi and intestinal contents without interference, with the recoveries of 85.24%~110.49% and the relative standard deviations (RSDs) of 2.63%~9.50% (Tables 2 and 3). The RSD was 1.06% when the same sample was repeated 6 times. The method is suitable for the determination of astaxanthin in biological samples with good spiked recovery and high accuracy.

 

2.2 Variation of astaxanthin content in biological samples

2.2.1 Changes of astaxanthin content in the intestinal contents of rats in different experimental groups    

The changes of astaxanthin content in the intestinal contents of rats are shown in Figure 2. The astaxanthin content in the intestinal contents of the rats in each experimental group showed an increase and then a decrease, reaching a peak at 2-4 h after the administration of the test substance by gavage, and then decreasing gradually, with very little astaxanthin detected in the intestinal contents at 12 h, and then the test substance was basically emptied out of the gastrointestinal tract at 24 h. At 0.5, 1, 2, 4, 8, and 10 h, the astaxanthin content in the intestinal contents of different experimental groups increased and then decreased. At 0.5, 1, 2, 4, 8 and 10 h, the astaxanthin content in the intestinal contents of rats in different experimental groups showed significant differences (P<0.05). In the astaxanthin group, astaxanthin peaked at 2 h (18.1±2.59 μg), in the Antarctic krill oil+astaxanthin group, astaxanthin peaked at 4 h (64.9±9.54 μg), and in the fish oil+astaxanthin group, astaxanthin peaked at 2 h (57.5±4.98 μg).

 

2.2.2 Changes of astaxanthin content in rat small intestine villi in different experimental groups    

The changes of astaxanthin in rat small intestinal villi are shown in Figure 3. The astaxanthin content in rat small intestinal villi gradually increased after gavage, reached the peak at 4 h, and then gradually decreased, and was basically undetectable at 16 h. At 1, 2, 4, 8 and 10 h, there were significant differences (P<0.05) between different experimental groups in astaxanthin content in rat small intestinal villi. At 1, 2, 4, 8 and 10 h, the astaxanthin content in the small intestine villi of rats in different experimental groups showed significant differences (P<0.05). In the astaxanthin group, the peak astaxanthin content in rat small intestinal villi was (101.8±25.52) ng; in the fish oil+astaxanthin group, the peak astaxanthin content in rat small intestinal villi was (226.7±41.39) ng, which was 2.23 times higher than that in the astaxanthin group (P<0.05); in the Antarctic krill oil+astaxanthin group, the peak astaxanthin content in rat small intestinal villi was (700.2±128.68) ng, which was 2.23 times higher than that in the astaxanthin group (P<0.05). (128.68)ng, 6.88 times higher than that in the astaxanthin group (P<0.05), and 3.09 times higher than that in the fish oil+astaxanthin group (P<0.05).

 

2.2.3 Changes of astaxanthin content in serum of rats in different experimental groups

The changes of astaxanthin in serum of rats are shown in Fig. 4. The serum astaxanthin content of rats in each experimental group started to increase slowly after 0.5 h of gavage administration, reached a peak at 8 h, and then began to decline gradually, returning to the initial level at 12-16 h. The serum astaxanthin content of rats in each experimental group was significantly different from that of rats in the 0.5, 1, 2, 4, 8, 10, and 12 h time points. The serum astaxanthin content of rats in different experimental groups differed significantly (P<0.05) at the time points of 0.5, 1, 2, 4, 8, 10 and 12 h. The serum astaxanthin content of rats in the astaxanthin group increased slowly after 0.5 h of gavage administration. The peak serum astaxanthin content of rats in the astaxanthin group was (1.89±0.26) μg/L; the peak serum astaxanthin content of rats in the fish oil+astaxanthin group was (3.94±0.27) μg/L, which was 2.08 times higher than that of the astaxanthin group (P<0.05); the peak serum astaxanthin content of rats in the Antarctic krill oil+astaxanthin group was (15.8±2.09) μg/L, which was 8.37 times higher than that of the astaxanthin group. The peak serum astaxanthin content of rats in the Antarctic krill oil+astaxanthin group was (15.8±2.09) μg/L, which was 8.37 times higher than that of the astaxanthin group (P<0.05) and 4.02 times higher than that of the fish oil+astaxanthin group (P<0.05).

 

2.2.4 Evaluation of bioavailability The AUC0-t values of serum astaxanthin content in rats are shown in Table 4. The bioavailability of astaxanthin in rats of different experimental groups was significantly different (P<0.05). In the astaxanthin group, the AUC0-t value was (14.4±0.21) μg-h/L; in the fish oil+astaxanthin group, the AUC0-t value was (23.4±0.46) μg-h/L, which was 1.63 times higher than that in the astaxanthin group (P<0.05); and in the Antarctic krill oil+astaxanthin group, the AUC0-t value was (108.4±2.34) μg-h/L, which was 7.55 times higher than that in the astaxanthin group. The AUC0 -t value of rats in the Antarctic krill oil + astaxanthin group was (108.4 ± 2.34) μg-h/L, which was 7.55 times higher than that of the astaxanthin group (P<0.05) and 4.63 times higher than that of the fish oil + astaxanthin group (P<0.05).

 

3 Discussion

According to the current reports [14-15, 18, 23, 27-30], the digestion and absorption process of natural astaxanthin is presumed to be as follows: astaxanthin in the food matrix is gradually released by oral mastication, gastric peristalsis, and digestive enzymes, and the released astaxanthin (either in the free state or the esterified state) mixes with lipids and is encapsulated in lipid droplets in the stomach, and then enters the small intestine under the action of digestive enzymes, such as pancreatic lipase, isomerase, etc., astaxanthin is released from the lipid droplets, and the esterified state is simultaneously digested into the free state [23, 28-29], After entering the small intestine, astaxanthin is released from the lipid droplets by the action of digestive enzymes such as pancreatic lipase, isomerase, etc. Astaxanthin in the esterified state is simultaneously digested to the free state [23, 28-29], and then emulsified with fatty acids, monoacylglycerides, phospholipids, bile salts, etc. in the system to form mixed micelles; subsequently, mixed micelles encapsulating free astaxanthin are absorbed into the epithelial cells of the small intestine by simple diffusion, passive diffusion, or cholesterol transporter [30]. Subsequently, the free astaxanthin micelles are absorbed by small intestinal epithelial cells by simple diffusion, passive diffusion or cholesterol transport[30] . Inside the small intestine, astaxanthin is encapsulated in celiac particles and enters the lymphatic system, then the blood circulation, and eventually reaches target organs such as the liver, heart, and spleen [31]. Red algae are currently recognized as the preferred food ingredient for natural astaxanthin, and their astaxanthin content is mainly in the esterified form, with a small amount of astaxanthin in the free form [14, 23].

 

In the present study, the astaxanthin content in the intestinal contents of rats peaked at 2-4 h, in the villi of the small intestine at 4 h, and in the serum at 8 h after the administration of astaxanthin extract by gavage, and the dynamics of digestion and absorption were basically consistent with the results of the studies conducted by Zhou Q. X. et al[23] and Coral-Hinostroza et al[28] .

Natural carotenoids generally have a low utilization rate for digestion and absorption [15, 17], and the addition of lipids to the intake process is an effective way to improve the situation [18-20, 32]. In the present study, the administration of dietary lipids of marine origin, such as Antarctic krill oil or fish oil, to rats by gavage with astaxanthin extracts from the red algae Rhodophyta rainforestris, resulted in a significant increase in the serum astaxanthin level and the AUC0-t value of the rats, which indicated that the Antarctic krill oil or fish oil significantly improved the bioavailability of the natural astaxanthin in the rats. In the in vivo digestive environment, physical, chemical, biological and other factors, the effect of food matrix components on the molecular conformation, solubility, stability of carotenoids, is the main reason for the changes in the nutritional properties of carotenoids [32]. In the present study, the detection of astaxanthin in the intestinal contents of rats in both the Antarctic krill oil + astaxanthin group and the fish oil + astaxanthin group was significantly higher than that in the astaxanthin group, and it was hypothesized that the effects of Antarctic krill oil and fish oil in improving astaxanthin bioavailability were closely related to their ability to increase the solubility of astaxanthin and its stability in the gastric and intestinal tracts. In addition, it has been suggested that different types of dietary lipids have different effects on the bioavailability of carotenoids [19, 33-34].

 

The fish oil used in this study was triglyceride-type fish oil, and triglycerides are digested into monoacylglycerides under the action of pancreatic lipase in vivo, which can increase the micellization rate of carotenoids [34], and promote the effective absorption of carotenoids by small intestinal epithelial cells. This conclusion is supported by the results of astaxanthin content changes in rat small intestinal villi in the present study. It is interesting to note that Antarctic krill oil is more effective in improving astaxanthin bioavailability than triglyceride fish oil. In addition to triglycerides, the content of phospholipids in Antarctic krill oil reaches more than 40%, and phospholipids are important structural substances that emulsify and form mixed micelles by wrapping around other dietary components during the digestive process of the body, and have been well established in building micellar transport systems to improve the bioavailability of fat-soluble dietary functional factors, insoluble pharmaceuticals, etc. [35-37]. The high phospholipid content of Antarctic krill oil is a nutritional characteristic that determines its superiority to fish oil in improving astaxanthin bioavailability, and this nutritional characteristic will also provide great scope for the development of other derivatives based on Antarctic krill oil.

 

4 Conclusion

Lipids of marine origin, such as Antarctic krill oil or fish oil, can significantly improve the bioavailability of astaxanthin of red algae origin in rats, with Antarctic krill oil being more effective. Antarctic krill oil to improve the bioavailability of natural astaxanthin mechanism mainly includes: on the one hand, Antarctic krill oil can improve the solubility of astaxanthin and its stability in the stomach and intestinal tract; on the other hand, the Antarctic krill oil contains triglycerides and a high content of phospholipids, digestive enzymes digested by the organism can increase the rate of astaxanthin microcellulose, to promote the small intestinal epithelial cells for its effective absorption. These two mechanisms deserve to be investigated and confirmed. The results of the present study indicate that the development of functional formulae of Antarctic krill oil and astaxanthin is feasible, and will effectively guide the development of Antarctic krill oil-related derivatives, which is of great significance in ensuring the development of the Antarctic krill industry in China.

 

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