Transepithelial Transport Mechanisms Of 7,8-dihydroxy-flavone In Human Intestinal Caco-2 Cells Model And Its Ameliorative Bioavailability Based On Nanotechnology

7,8-dihydroxyflavone(7,8-DHF),a naturally occurring flavone monomeric compound in Godmania aesculifolia and Tridax procumbens,was identified to act as a high-affinity Trk B receptor agonist that mimicked physiological actions of brain-derived neurotrophic factor(BDNF)downstream signaling,such as MAPK/ERK,PI3K/Akt and PCγ1.Numerous evidences have confirmed that 7,8-DHF attenuated many BDNF-involved human disorders such as Alzheimer’s disease,Parkinson’s disease,depression,and obesity.Nevertheless,sulfation,glucuronidation and methylation of the catechol group in the liver caused its oral bioavailability extremely low(only 4.6%).The oral bioavailability of the prodrug(R13)modified with chemical method was increased to 10.5%,but this method was not applicable to the development of functional food.Based on the understanding of 7,8-DHF digestion and absorption,this paper focused on the physical modification means of nanotechnology,and selected different foodborne biological macromolecules to optimize the construction of nanoparticles,so as to significantly improve the oral bioavailability of 7,8-DHF.First,7,8-DHF was investigated by using Caco-2 cells model to explore its transepithelial mechanism.The ameliorative effect of natural dietary flavonoids on transport efficiency of7,8-DHF were also evaluated.Then,based on zein,supplemented by protein(lactoferrin,LF),surfactant(sophorolipid)and polysaccharides(sodium alginate and carboxymethyl cellulose sodium),binary and ternary nanoparticles were fabricated by anti-solvent co-precipitation(ASCP)method.Their physicochemical properties,structural characteristics and system stability were evaluated.Finally,structural characterization,storage stability,anti-digestibility in vitro and oral bioavailability of7,8-DHF loaded binary and ternary nanoparticles were systematically studied.The main research contents and results are as follows:1.The transport mechanisms of 7,8-DHF by using the Caco-2 cells model to determine the bi-directional permeability at different environmental factors(time,concentration,pH and metabolic inhibitors etc).The influx and efflux characters of7,8-DHF were also clarified.7,8-DHF was poorly transported across Caco-2 cell monolayers by mainly passive diffusion via a transcellular pathway The transport of7,8-DHF was time and concentration-dependent in both apical(AP)to basolateral(BL)side and the reverse direction.Interestingly,decreasing the pH from 7.4 to 6.0markedly enhanced 7,8-DHF transport.It is noteworthy that 7,8-DHF transport was strongly inhibited by metabolic inhibitors.The efflux ratio(ER)values at different concentrations were all above 1.5,indicating the existence of efflux transporter.We found breast cancer resistance protein(BCRP)was not involved in 7,8-DHF secretion,and the transport mechanism of 7,8-DHF is passive transport with active efflux mediated by P-glycoprotein(P-gp)and multidrug resistance-associated proteins(MRPs),particularly MRP2.Moreover,the use of various influx transporter inhibitors in Caco-2 cells showed that organic cation transporters(OCTs)and organic anion-transporting polypeptides(OATPs)participated in 7,8-DHF transport.2.The natural vegetal flavonoids,potential P-gp inhibitors,were selected to promote the transport efficiency of 7,8-DHF in Caco-2 cells model.And the effect of dietary flavonoids on the stability of 7,8-DHF in in vitro gastrointestinal digestion was also evaluated.The results showed that quercetin and kaempferol could improve the transport efficiency of 7,8-DHF in Caco-2 cells model.Moreover,the structure of7,8-DHF could maintain stability in in vitro digestive system,while quercetin and kaempferol did not affect its structural stability.3.Stable zein/lactoferrin(LF)binary nanoparticles were successfully fabricated by ASCP method to encapsulate 7,8-DHF.The results showed that the mean particle size of zein/LF binary nanoparticles was about 74 nm with a low polydispersity index(PDI<0.200)and turbidity(<0.300)values.Zein/LF binary nanoparticles had good stability against aggregation at a wide pH range of 3.0～9.0,ion strengths(0～500mmol/L Na Cl)at neutral pH and long-term storage.The encapsulation efficiency(EE)of zein/LF composite nanoparticles(98.31%)was significantly higher than zein nanoparticles(46.38%).The freeze-dried 7,8-DHF loaded zein/LF nanoparticles had an excellent water redispersibility with high EE(92.66%)and low PDI value(0.265).Fluorescence quenching revealed both LF and zein could interact with 7,8-DHF via hydrophobic interaction and the number of binding sites was 1.23 and 1.29,respectively.Transmission electron microscopy(TEM)and scanning electron microscope(SEM)displayed nanoparticles were nanoscale spherical shape and LF changed surface morphology of unloaded and loaded zein nanoparticles.X-ray diffraction(XRD)indicated 7,8-DHF was presented in an amorphous state inside the nanoparticles.Fourier-transform infrared spectroscopy(FTIR)analysis showed effective hydrogen bonding and hydrophobic effects existed between zein and LF,while electrostatic interaction,hydrogen bonding and hydrophobic interactions existed between zein and 7,8-DHF.In order to further improve the stability of zein/LF binary nanoparticles,three different molecular weights(10,40 and 70 k Da)of dextran were used to graft LF through Maillard reaction to form glycosylated LF.Then zein/glycosylated LF binary nanoparticles were built.Three zein/glycosylated LF binary nanoparticles showed small particle sizes(78～87 nm)with low PDI(<0.230)and turbidity(<0.220)values.Zein/glycosylated LF binary nanoparticles showed strong stability to ionic strength(0～500 m M Na Cl)at pH 3.0～9.0 compared to zein/LF binary nanoparticles.The presence of glycosylated LF further improved the encapsulation efficiency(EE)of7,8-DHF(above 98.50%).Differential scanning calorimetry(DSC)showed zein/glycosylated LF binary nanoparticles had good thermostability compared to zein and zein/LF nanoparticles.FTIR analysis confirmed more effective hydrogen bonding and hydrophobic effects were formed between zein and glycosylated LF.4.7,8-DHF loaded in zein/-sophorolipid nanoparticles with two blending sequences of sophorolipid(DHF-Z/S and DHF-Z-S)were fabricated.The results showed that blending sequences significantly affect the physicochemical properties of nanoparticles,DHF-Z-S nanoparticles had a smaller particle size,lower polydispersity index and turbidity,higher negative charge,entrapment efficiency and loading capacity compared to DHF-Z/S nanoparticles.TEM and SEM displayed DHF-Z-S and DHF-Z/S nanoparticles were nanoscale core-shell spherical shape and sophorolipid changed surface morphology of zein nanoparticles.FTIR and fluorescence spectrum analysis confirmed effective hydrogen bonding,electrostatic interactions and hydrophobic effects were formed among 7,8-DHF,zein and sophorolipid and the forming stronger hydrogen bonding and hydrophobic effects of DHF-Z-S nanoparticles were found.The encapsulated 7,8-DHF was in an amorphous state(rather than crystalline form)as detected by XRD.Circular dichroism(CD)revealed that 7,8-DHF and sophorolipid were capable of remarkably changing the secondary structure of zein.Zein-sophorolipid-polysaccharide ternary nanoparticles were fabricated by antisolvent co-precipitation method.The results showed that the mass ratio of zein and polysaccharides and polysaccharide type significantly affect the physicochemical properties of the nanoparticles,including particle size,polydispersity index,zeta potential and turbidity.Sodium alginate(ALG)and sodium carboxymethyl cellulose(CMC)enhanced the entrapment efficiency of 7,8-DHF in zein-sophorolipid nanoparticles.The presence of ALG and CMC solved the precipitation phenomenon of zein/sophorolipid nanoparticles at low pH value.FTIR confirmed effective hydrogen bonding,electrostatic interactions and hydrophobic effects were formed among zein,sophorolipid and polysaccharide.CD revealed that the addition of CMC and ALG had no apparent impact on secondary structure of zein in the 7,8-DHF loaded zein-sophorolipid nanoparticles.DSC showed the addition of CMC and ALG had good thermostability compared to DHF-Z-S nanoparticles.TEM and SEM displayed nanoparticles were nanoscale spherical shapes(above 150 nm)and CMC or ALG changed surface morphology of DHF-Z-S.5.The storage stability,gastrointestinal stability,in vitro bioaccessibility and in vivo bioavailability of 7,8-DHF loaded zein binary and ternary nanoparticles were investigated.The results showed that 7,8-DHF loaded zein binary and ternary nanoparticles both enhanced the stability of 7,8-DHF during storage.Meanwhile,zein/glycosylated LF binary nanoparticles and zein-sophorolipid-polysaccharide ternary nanoparticles had a certain resistance to gastric juice and intestinal juice in in vitro digestion system,which ensured the structural stability of nanoparticles.SEM also found that after digestion,although the average particle size increased,they could still maintain a certain spherical shape.Furthermore,they improved in vitro bioaccessibility of 7,8-DHF(Increased by 1.76～4.65 times).In vivo bioavailability studies confirmed that zein/glycosylated LF binary nanoparticles and zein-sophorolipid-polysaccharide ternary nanoparticles could both increase the bioavailability of 7,8-DHF(The relative bioavailability increased by 1.76～4.65 times)and prolonged its retention time in the blood.In summary,the transport mechanisms of 7,8-DHF might be the passive facilitated diffusion pathway mediated by energy-dependent efflux proteins and influx proteins.Zein/glycolated LF binary nanoparticles and zein-sophorolipidpolysaccharide ternary nanoparticles had good physicochemical stability and gastrointestinal stability,and improved the storage stability,in vitro bioaccessibility and in vivo bioavailability of 7,8-DHF.The raw materials used in this process are all foodborne ingredients,and the organic solvent involved in this process is only edible alcohol(ethanol).The natural source of the raw materials and the greening process can ensure the safety of 7,8-DHF composite nanomaterials.Thus,in this paper,nanotechnology showed a very broad application prospect on development of functional food,especially for such functional factor as flavonoids and phenolic acids.