The absorption kinetics of Antarctic krill oil phospholipid liposome in blood and the digestive tract of healthy mice by single gavage

Lin Li,Chengheng Wng,Shn Jing,Rong Li,Tintin Zhng,Chnghu Xue,b,Teruyoshi Yngit,Xioming Jing,*,Yuming Wng,b,*

a College of Food Science and Engineering,Ocean University of China,No.5 Yushan Road,Qingdao,266003,PR China

b Laboratory for Marine Drugs and Bioproducts,Pilot National Laboratory for Marine Science and Technology,Qingdao,266237,Shandong Province,PR China

c Laboratory of Nutrition Biochemistry,Department of Applied Biochemistry and Food Science,Saga University,Saga,840-8502,Japan

Keywords:

ABSTRACT

1.Introduction

Antarctic krill(Euphausia superba),an important marine crustacean organism,exhibits large biomass in the world.Antarctic krill has been gaining increasing interest worldwide for its nutritional value and potential utilization as human food[1].Antarctic krill oil(AKO)is an important ingredient rich in eicosapentaenoic acid(EPA)and docosahexaenoic acid(DHA)with various functional properties.Dietary administration with 1% AKO could effectively ameliorate learning and memory deficits and ease the anxiety in SAMP8 mice.Furthermore,Antarctic krill oil could alleviate articular cartilage degenerationviaactivating chondrocyte autophagy and inhibiting apoptosis in osteoarthritis mice[2].Antarctic krill oil also could improve glucocorticoid-induced osteoporosisviapromoting bone formation[3].In addition,dietary AKO was reported to regulate lipid and glucose metabolism and energy expenditure by suppressing high fat diet-induced fat accumulation with a decrease of malondialdehyde content and increase of superoxide dismutase content[4].Dietary AKO also could suppress the development of autoimmune murine lupus by regulating inflammatory mediators in an organ-specific manner,thereby prolonging life span[5].Moreover,some recent studies showed that supplementation with AKO could alter microbiota and thus help to alleviate brain-aging,hyperlipidemia and obesity[6-8].

Antarctic krill oil exhibits a diverse profile of lipid classes including phospholipids(PLs),triacylglycerols(TAGs),diacylglycerols(DAGs),monoacylglycerols(MAGs)and free fatty acids(FFAs)[9].The lipid compositions of krill oil are usually influenced by many factors,such as seasonal variation,interannual environmental changes,capture stations,storage conditions,etc.Krill oil is characterized by a high PL content about 50%,meanwhile it contains 30%-40% TAG depending on sample variety and analysis method[10,11].Various studies have showed that EPA and DHA in the form of phospholipids exert superior bioactivity to other forms[12-19].Functional beverage is one of the common forms in the nourishment market.Lipids usually cannot be directly developed into functional beverages due to their fat soluble property.A relatively stable dispersion in water for lipids usually require a lot of emulsifier.Furthermore,the obtained emulsion possesses larger size and is difficult to be absorbed.Interestingly,phospholipids can be made into liposomes without emulsifiers due to its amphiphilic characteristics[20].Liposomes can prolong functional components persistence in the body,which has been wildly applied in food sectors and pharmaceutical industry[21,22].

There is an inextricable link between the unique bioactivity and digestion and absorption.The digestion and absorption of TAGs and PLs are in different ways in the small intestine.TAGs is digested and absorbed under the emulsification of bile salts,while PLs can spontaneously form micelles and be transported in an aqueous environment[23].Fatty acids are usually released from sn-1 and sn-3 positions of the ingested TAGs under the action of pancreatic lipase[24].PLs are mainly hydrolyzed by pancreatic phospholipases A2(PLA2)to release the fatty acid from the sn-2 position and form the derived lyso-phospholipids[25].After the released fatty acids and lyso-phospholipids are taken up into enterocyte,some of the lyso-phospholipids is further hydrolyzed by an enterocyte-derived lysophospholipase into a fatty acid and glycerophosphocholine[26].However,the fate of various lyso-phospholipids with different fatty acid species has not been reported.At present,the specific digestion and absorption mechanisms of dietary phospholipids enriched with DHA/EPA are unclear.The absorption kinetics of Antarctic krill oil phospholipid liposome in vivo has not been elaborated,which restrict the molecular mechanism analysis related to its distinct bioactivities.Therefore,the aim of the present study was to investigate the digestion and absorption characteristics of Antarctic krill oil phospholipids(AKOP)in blood and the digestive tract of healthy mice by single gavage of phospholipids in liposome form.

2.Material and methods

2.1.Chemicals and reagents

The powder of Antarctic krill(Euphausia superba)was provided from the Dalian Marine Fisheries Group Corporation(Dalian,Liaoning,China).Internal standard(C15:0)was purchased from Nu-Chek Prep Inc.,(Elysian,MN,USA).Fatty acid methyl ester standard was purchased from Sigma-Aldrich.Methanol,acetone and chloroform were purchased from Sinopharm Chemical reagent Co.Ltd(Shanghai,China).Hexyl hydride was purchased from Tianjin Fuyu Fine Chemical Co.,Ltd(Tianjin,China).

2.2.The preparation of Antarctic krill oil phospholipids and fatty acid compositions analysis

The AKO was extracted from Antarctic krill(E.superba)according to the previous Folch method with minor modification[27].Briefly,the homogenate was prepared by placing the krill powder in a solution of chloroform:methanol(V/V=2:1)at the ratio of 1:8(m:V)for 5 h to extract lipids.The extraction was then mixed with water and a separatory funnel was used to thoroughly clear the chloroform phase.The chloroform layer was collected and evaporated to dryness under vacuum to obtain total Antarctic krill oil.The cold acetone was used to remove the TAGs for two times to obtain Antarctic krill oil phospholipids.All the procedures of lipid extraction were repeated for 3 times.The purity and content of AKOP was determined by thin layer chromatography(TLC)using neutral developing solvent(hexane,ether,and acetic acid;85:15:1)and phospholipid-molybdenum blue complex in hexane,respectively.The molecular class of phospholipids was confirmed by Waters 1525 Liquid Chromatograph with an Evaporative Light-scattering Detector using a ZORBAX Rx-SIL column(250 mm×4.6 mm×5 μm).The mobile phase A was hexyl hydride:isopropanol:5 mmol/L ammonium formate=82:17:1.0(V/V)and mobile phase B was isopropanol:water:5 mM ammonium formate=85:14:1.0(V/V).The flow rate was 1 mL/min,the column temperature was 35°C.The drift tube temperature was 60°C and the pressure of nebulizing gas was 25 psi.The elution procedure was shown in the Table 1.The main fatty acids were determined by Agilent 6890 Gas Chromatography with a flame-ionization detector using a HP-INNOWax capillary column(30 m×0.32 mm×0.25 μm).

Table 1 The elution procedure of HPLC-ELSD.

2.3.The preparation of AKOP liposomes

The liposomes of AKOP was prepared referring to the previously reported method[28].Concisely,phospholipids were dissolved in saline and preliminarily mixed to obtain phospholipid solution.Ultrasonic cell fragmentation apparatus was used to make fragmentation of phospholipid solution with the output power of 100 W for 10 min.The liposome dispersion eventually turned from milky to opalescent.The liposomes should be prepared daily before use.

2.4.Particle size and polydispersity index(PDI)measurements of AKOP liposomes

The particle size measurement of the freshly prepared AKOP liposomes was evaluated on a commercial laser light scattering instrument at 25°C with a 90°scattering angle.The polydispersity index(PDI)reflecting the particle size distribution of liposomes was also reported.

2.5.Animals experiment

Forty-two male Balb/c mice weighting(22±2)g were purchased from the Vital River Laboratory Animal Technology Co.,Ltd(Beijing,China).The mice were housed individually with food and water ad libitum throughout the feeding experiment in a room maintained at a 12-h light/dark cycle and a constant temperature of(22±2)°C.All aspects of the experiment were conducted according to guidelines provided by the ethical committee of experimental animal care at Ocean University of China(Qingdao,China).The mice were randomly divided into six groups,corresponding to the six time points in the course of digestion and absorption

All mice were supplied with ordinary diet which was based on AIN93 recipe.The compositions of diets were summarized in Table 2.All mice were fasted and given free water for 10 h before gavage.The gavage volume of the phospholipid liposome was calculated according to the EPA+DHA gavage amount of 4.70 mg.After oral administration with AKOP liposomes,mice in the corresponding group were sacrificed at 0,0.5,1,2,3 and 5 h,and blood was collected.Small intestinal contents were washed out with physiological saline and the wall of small intestinal was weighted and collected.The serum was extracted by centrifugation at 7500g for 15 min at 4°C.All fresh samples were stored at-20°C until analysis.

Table 2 Composition of Experimental Diet(g/kg).

2.6.Lipid extraction

Lipids in serum and small intestinal content were extracted according to the Bligh & Dyer method,and the recovery of lipid extraction was nearly 99%[29].In brief,chloroform and methanol were added to serum at the ratio of serum:chloroform:methanol=0.8:1:1(V/V)and then mixed.The mixture was centrifuged at 8000 r/min for 2-3 min,and the lipid was obtained from the lower layer of chloroform.Lipid in the wall of small intestinal was extracted according to Folch method,and the recovery of lipid extraction was nearly 99%[27].In brief,the wall of small intestine was extracted with chloroform:methanol:water(2:1:0.4)and placed into a separatory funnel for 24 h to thoroughly clear the chloroform phase.The chloroform layer was collected and evaporated to dryness under vacuum to obtain the lipid sample.The lipid was dissolved in petroleum ether,and stored at-20°C until analysis.

2.7.Analysis of fatty acid composition by gas chromatograph

The extracted lipids were then treated with methyl ester.Briefly,the lipids were prepared by transmethylation with a mixed solution of methanol and hydrochloric acid(HCL)(5:1,V/V)by shaking at 90°C for 3 h.C15:0was used as the internal standard.The lower limit for the detection of fatty acids was about 3.1-9.5 μg/mL.The main fatty acids were determined by Agilent 6890 Gas Chromatography with a flame-ionization detector using a HP-INNOWax capillary column(30 m×0.32 mm×0.25 μm).The temperature of the detector and injector was kept constant at 250°C and 240°C,respectively.The initial oven temperature was 170°C,held for 5 min,and then increased to 240°C at 3°C/min,kept for 30 min.The entire process lasted for 48.3 min and the balance time was 3 min.Nitrogen was used as carrier gas at the flow rate of 1.2 mL/min.

2.8.Statistical analysis

Statistical analysis was performed using the SPSS software.Oneway analysis of variance was used to evaluate the differences.All data were expressed as mean±SEM(standard error of the mean,indicated by error bars).The difference was considered significant when P＜0.05.

Fig.1.The lipid composition of Antarctic krill oil(AKO)and Antarctic krill oil phospholipid(AKOP)by TLC analysis.

Table 3 Main fatty acid composition of Antarctic krill oil phospholipids(AKOP)(n=3).

3.Results anddiscussion

3.1.The composition of AKOP

In order to determine the purity and content of the obtained Antarctic krill oil phospholipid,TLC analysis and colorimetric Method were carried out.As shown in Fig.1,there was almost no band of TAG on the TLC plate after cold acetone cleaning,suggesting that most TAG was removed from Antarctic krill oil.The content of phospholipid increased from 50.41%in the Antarctic krill oil to 82.73%in the obtained AKOP.The major molecular type was phosphatidylcholine,which was confirmed by HPLC-ELSD(Fig.2).The contents of the DHA and EPA in AKOP were 19.63%and 9.68%,respectively(Table 3).

3.2.Characterization of the AKOP liposome

Particle size and PDI are important parameters to prepare stable liposome targeting nutritional and medical applications.The average particle size diameter of the AKOP liposome by dynamic laser light scattering was(73.7±1.0)nm(Fig.3).The PDI value was 0.171,showing acceptable homogeneity.

Fig.2.The major molecular type of Antarctic krill oil phospholipid liposome determined by HPLC-ELSD.PC:phosphatidylcholine.

Fig.3.The particle size of Antarctic krill oil phospholipid liposome evaluated on commercial laser light scattering instrument at 25 °C with a 90° scattering angle.

Table 4 The fatty acid composition in serum within 5 h after administration of AKOP liposome(n=7).

3.3.The fatty acid composition in serum within 5 h after administration of AKOP liposome

The dietary phospholipids are mainly hydrolyzed into fatty acid and lyso-phospholipids in the small intestine by pancreatic phospholipases A2.These hydrolysis products are absorbed by intestinal enterocytes and re-esterified to form chylomicrons,which will be secreted into the lymphatic system and enter circulation[30].As is known that the first 5 h after dietary consumption is considered as an important phase for digestion and absorption of lipids.Therefore,we determined the fatty acids composition in serum within 5 h after administration of the AKOP liposome to understand its digestion and absorption process in vivo by collecting the serum samples at 0,0.5,1,2,3 and 5 h.As shown in Table 4,the level of main serum fatty acid,such as C16:0and C18:1,showed a peak at 0.5 h,and then continued to decreased up to 5 h.The levels of C16:0and C18:1increased by approximately 1.9-fold and 3.1-fold,respectively,at 0.5 h in comparison with 0 h,which was consistent with the high content of C16:0 and C18:1 about 32.29%and 18.02%in the AKOP.DHA and EPA are considered as important fatty acids in AKO.Therefore,we depicted the time profile of DHA content showed in Fig.4.The result showed that the content of DHA in serum decreased after increasing to 0.2 mg/mL at 0.5 h(P＜0.0001,compared to 0 h),and then continued to rise with a peak at 2 h.Notably,the level of EPA in the serum was too low to detect as the lower limit for the detection of fatty acids was about 3.1-9.5 μg/mL,which suggested that EPA might be retained in lymph or metabolized into other fatty acids derivatives,such as DHA,before entering the blood.Michael et al.found that the ratio of EPA to DHA in total lymph lipids was changed(about DHA:EPA=9.5:13)at 4 h after administration of TAG(DHA:EPA=9.2:10.2)in rat,which suggested that lymph had a stronger retention effect on EPA than DHA[31].In addition,it has been reported that EPA could convert into DHA in vivo[32].The present data showed that the DHA in serum peaked at 2 h and then decreased after administration of AKOP liposome as a result of transport into other organs and metabolism.010000030404010101

Table 5 The main fatty acid(FA)composition of total lipids in small intestinal content within 5 h after administration of AKOP liposome(n=7).

Fig.4.The DHA level(mg/mL)in serum within 5 h after administration of Antarctic krill oil phospholipid liposome.All data were presented as mean±SEM(n=7).Different letters indicated significant difference at P＜0.05 among timepoints.

3.4.The fatty acid composition in small intestinal content within 5 h after administration of AKOP liposome

In order to illustrate the digestion and absorption process of AKOP liposome,we further determined the fatty acid composition in small intestinal content within 5 h after administration of AKOP liposome.The results showed that the main fatty acids,such as C14:0,C16:0,C16:1,C18:1,EPA and DHA,in intestinal content exhibited a tendency with very steep initial increase reaching a maximum at 0.5 h(P＜0.0001,compared to the other timepoints tested)after oral administration of AKOP liposome,and then continued to decreased up to 5 h(Table 5).As shown in the Fig.5,EPA and DHA in the intestinal content were reduced by 65.5% and 61.9%,respectively,from 0.5 h to 1 h,and then remained stable.Some studies have demonstrated that the bioavailability of krill oil was greater than fish oil in form of TAG,which might be largely attributed to the presence of phospholipids[33,34].PL was more easily to bind with PLA2 and other hydrolyzing enzymes on account of its molecular structure,thereby accelerating the digestion of lipids[33,34].The present results showed that AKOP liposome was almost completely digested in the small intestine in 1 h.

3.5.The fatty acid composition in the wall of small intestine within 5 h after administration of AKOP liposome

Since the intestinal wall was an important part for the absorption of lipids into the blood,we determined the fatty acid composition in the wall of small intestine within 5 h after administration of AKOP liposome(Table 6).The DHA and EPA in the wall of the small intestine initially increased and reached the peak at 1 h,suggesting that the hydrolysis products had been absorbed by intestinal enterocytes.The reduction of fatty acids from 1 h to 2 h indicated that the absorbed fatty acids were efficiently reprocessed and transported,which was consistent with serum DHA level.It was worth noting that the content of DHA is much higher than that of EPA.In addition,EPA could be converted into DHA in vivo,which might be the reason for the high content of DHA[35,36].57346211804148

Fig.5.a.The total content(mg)of DHA in small intestinal content within 5 h after administration of Antarctic krill oil phospholipid liposome.b.the total content(mg)of EPA in small intestinal content within 5 h after administration of Antarctic krill oil phospholipid liposome.All data were presented as mean±SEM(n=7).Different letters indicated significant difference at P＜0.05 among timepoints.

Table 6 The main fatty acid(FA)composition(%)in the wall of small intestine within 5 h after administration of AKOP liposome(n=7).

4.Conclusion

In conclusion,the phospholipid was separated from Antarctic krill oil using cold acetone to remove triacylglycerols.The major type of the obtained Antarctic krill oil phospholipid was phosphatidylcholine,and the total contents of the DHA and EPA were 29.31%.The lipid analysis in serum,small intestinal content and wall was carried out after oral administration of Antarctic krill oil phospholipid liposome in the present study to illustrate its absorption kinetics in blood and the digestive tract of healthy mice by single gavage.Results showed that AKOP liposome was almost completely digested in the small intestine in 1 h and the hydrolysis products could be quickly absorbed by intestinal enterocytes.In addition,the DHA in serum peaked at 2 h after administration of AKOP liposome.The obtained results might provide a scientific basis for the molecular mechanism analysis related to distinct bioactivities of Antarctic krill oil phospholipid.

Funding

This work was supported by National Key R&D Program of China(2018YFD0901103),National Natural Science Foundation of China(31901688),Natural Science Youth Foundation of Shandong Province(ZR2019QC004),National Natural Science Foundation of China-Shandong Joint Fund for Marine Science Research Centers(U1606403),Laboratory for Marine Drugs and Bioproducts of Pilot National Laboratory for Marine Science and Technology(Qingdao,LMDBKF201807),Grant-in-Aid for Scientific Research(No.16K00890).

Availability of data and materials

All data generated or analyzed during the current study are available from the corresponding author on reasonable request.

Authors’contributions

LL,YMW,XMJ conceived and designed the experiments;LL,CCW,SJ,RL performed the experiments;LL,TTZ,CHX analyzed the data;CHX,TY,TTZ,YMW contributed reagents/materials/analysis tools;LL,XMJ wrote the paper.All authors have read and approved the final manuscript.

Ethics approval

Animal experiments described in our study were approved by the Ethical Committee of Food Science and Human Health Laboratory of Ocean University of China.

Declaration of Competing Interest

The authors declare that they have no competing interests.

Acknowledgements

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