Study On The Loading Of Flavonoids By Plant Ferritin And The Improvement Of Its Bioavailability

Baicalein and naringenin are flavonoid compounds,both of which have good antioxidant properties and nutritional value.However,their poor water solubility and low bioavailability limit their applications.Ferritin,as protein that stores iron and regulates iron balance,has hollow cage structure formed by self-assembly 24 subunits,which is widely distributed in animals,plants and microorganisms.Among them,plant ferritin is the main form of iron in plants,butwhich has a good effect of iron supplementation and will not cause toxic and side effects.Embedding the flavonoid compounds by using the cage nano structure of the novel plant ferritin is beneficial to improving the water solubility,stability and bioavailability of the embedding substances,and further widens the application of the plant ferritin.In addition,the double protective layers formed by chitosan modification on the surface of ferritin is beneficial to further protecting the stability of the embedding substance.In this work,plant ferritins from alfalfa(AFn)and green algae(Up Fer)were expressed in E.coli by genetic engineering,and the pure target proteins were obtained by further purification with tags.The flavonoids baicalein(BIC)and naringenin(NAR)were encapsulated into the plant protein cage by urea method to construct alfalfa ferritin-baicalein nanocomposites(AFn-BIC)and green algae ferritin-naringenin nanocomposites(Up Fer-NAR).In addition,chitosan-alfalfa ferritin-baicalein nanocomposite(CAFn-BIC)was prepared from the surface-modified chitosan of alfalfa ferritin-baicalein nanocomposite,and its antioxidant capacity,digestion resistance and bioavailability were evaluated.The results were as follows:(1)Two new plant ferritins,AFn and Up Fer,were successfully expressed and purified.AFn and Up Fer plant ferritin were hollow cage structures formed by self-assembly of 24 subunits,and the secondary structure was a typical α-helix,with particle sizes of 12.0 ± 0.3 nm and 12.1 ± 0.3 nm,respectively.AFn dissociated at p H 2 or p H 13,and self-assembled under neutral conditions,while Up Fer dissociated at p H 3 or p H 12,and self-assembled under neutral conditions.(2)AFn-BIC and Up Fer-NAR nanocomposites were successfully prepared by using low-concentration urea method and loading flavonoids into protein cages.The prepared AFn-BIC was modified with chitosan by chemical method to prepare CAFn-BIC nanocomposites.According to the calculation,each AFn and Up Fer protein cage could encapsulate 52 BIC and 265 NAR molecules,respectively.The prepared AFn-BIC and Up Fer-NAR nanocomposites were still 24-mer,and the secondary structure maintained the typical α-helix structure.The encapsulation of flavone compounds has no effect on the structure of ferritin.Particle sizes were 13.3 ± 0.3 nm and 13.5 ± 0.1 nm,respectively.Chitosan was bound to ferritin surface by electrostatic interaction.Fluorescence analysis showed that one ferritin molecule could be modified by eight chitosan molecules.The prepared CAFn-BIC nanoparticles had a particle size of 14.5 ± 0.3 nm.(3)The bioavailability of the prepared CAFn-BIC and AFn-BIC nanoparticles were evaluated.Due to the shielding effect of ferritin,the free radical scavenging ability of BIC encapsulated in protein cage was lower than that of free BIC,while the antioxidant activities were still retained.In the simulated gastric digestion experiment,the protective layer formed by chitosan modification on ferritin surface could improve the anti-digestion ability of AFn-BIC nanoparticles and reduced the release of BIC during digestion.Cytotoxicity test results showed that AFn protein carrier had no obvious toxicity to bovine mammary epithelial cells(BMECs),which proved that AFn protein carrier had high safety and biocompatibility.In Caco-2 cell absorption experiment,under the unique transport route of protein cage,both CAFn-BIC and AFn-BIC nanoparticles could promote the cell absorption of BIC,and the absorption amount of BIC was much higher than that of free group,indicating that the bioavailability of BIC was obviously improved.These results also showed that alfalfa ferritin could be used as an excellent nanocarrier to improve the bioavailability of insoluble biomolecules.