Bioavailability And Antagonistic Factors Of Fluoride In Dark Tea

Brick tea is made by old tea leaves,generally contains higher fluoride than other tea.In Northwest China,some minority nationalities have the habit of drinking a large amount of high fluoride dark tea,and excessive consumption of dark tea leads to fluorosis.The harm of fluoride in dark tea depends not only on the content of fluoride,but also on the bioavailability of fluoride.The chemical components of dark tea are complex,and the presence of these compounds may affect the bioavailability of fluoride in tea leaves.Besides,consumers are accustomed to drink dark tea supplement with milk powder,butter and salt,which also affect the bioavailability of fluoride in tea.Therefore,it is of great significance to study the bioavailability of fluoride in dark tea and screen the antagonistic factors of fluoride absorption in tea for the health of consumers and safety of tea drinking.In this dissertation,effects of dietary supplementation on the bioavailability of fluoride in dark tea were evaluated in animal experiments,and Caco-2 cell line was carried out to screen the antagonistic factors affecting fluoride absorption and investigate its mechanism.The details were summarized briefly as follows:(1)NaF solution(NaF),dark tea infusion(DTS)and dark tea infusion supplement with milk powder,butter and salt,contained 45 mg/L fluoride,were administrated to rats.Blood samples were collected at different time points after oral administration,fecal were collected during experiment.The amount of fluoride in plasma and fecal samples were determined using ion electrode method,and the pharmacokinetic parameters were evaluated.The results showed that the area under the plasma curve(AUC0-t)and maximum plasma concentrations(Cmax)of DTS were decreased 41.10% and 45.78% respectively,compared with Na F,indicated the bioavailability of fluoride in dark tea were lower than Na F solution.With the supplementary of milk powder,the bioavailability of fluoride in dark tea was significantly declined,the AUC0-t decreased 24.44%,73.33% and 89.78%with addition of low,medium and high dosing of milk powder.A increase amount of milk powder supplementation implied significantly increase of peak time(Tmax),the highest dosing of milk powder supplementation lead to a 6.37 times increase of Tmax.T1/2 was reduced by addition of milk powder,which suggests milk powder increase the eliminate rate of fluoride in dark tea.Milk powder also increased the amount of fluoride in fecal.Comparing with DTS,AUC0-t showed no statistical differences with addition of butter,suggested butter does not affect the bioavailability of fluoride in dark tea.However,butter significantly decrease the Cmax of fluoride in dark tea,and Tmax was increased with the addition of low dosing of butter,while medium dosing of butter lead to shorter T1/2,which indicate butter increase the eliminate rate of fluoride in dark tea.The bioavailability of fluoride in dark tea was related to the dosage of salt,0.4% salt significantly increase bioavailability of fluoride in dark tea,while 2% salt has no significant effect,compared with DTS.(2)Caco-2 cells were cultured for 21 days in Transwell,morphological characteristics of the cell membrane and transepithelial electrical resistance(TEER)were evaluated.The results showed that the TEER of Caco-2 cell model reached 863.95±46.70 ?·cm2 after 21 days,and had formed integrate brush border microvillus and tight junction,suggested that cell monolayer was established successfully and appreciated to study the absorption and transport of compounds in vitro.(3)Fluoride in dark tea infusion was separated with resin,and investigated transport of two different species of fluoride with Caco-2 cell models.The results showed that fluoride in cation species lead to a decrease transport from apical to basolateral side(AP-BL),while no remarkable effect was observed in contrary direction,compared with Na F solution.The transportation of anionic fluoride is similar to Na F solution in both directions.In conclusion,fluoride in cation species may lead to lower bioavailability in dark tea infusion.(4)Caco-2 cell models were carried out to investigate the effect of Al3+,EGCG and Na2 SeO3 on transport and intracellular accumulation of fluoride.The results showed that Al3+ increased intracellular accumulation of fluoride significantly,the amount of accumulation was related with Al3+ concentration.2.5 mg/L,10 mg/L and 40 mg/L Al3+decreased the transport of fluoride in both direction,moreover,10 mg/L Al3+obtained maximum effect.This might because of the species of fluoride in HBSS had changed after the addition of Al3+,and the mechanisms of accumulation and transport of different forms of fluoride in Caco-2 cells may be different.Only the maximum addition of Na2 SeO3declined the transport of fluoride.10,20,40 ?M EGCG had no significant effect on transport of fluoride in both direction,while 100 ?M EGCG inhibited the transport of fluoride from apical to basolateral side.(5)The transport and cellular accumulation of fluoride with addition of 2.5 mg/L,10 mg/L and 40 mg/L Al3+ were investigated in Caco-2 cell models in various conditions(temperature,uncoupler,EDTA).The results showed that 4 °C and Na N3 inhibited the transport and accumulation of fluoride ion(F-),suggesting that active transport may involve in transport and accumulation of F-.Opening of tight junction with EDTA increased the transport of F-,indicating F-transport by paracellular pathway.Therefore,active tranport participate in accumulation of F-,and its transport takes place mainly by passive diffusion across cell junctions and active transport.The accumulation and transport of fluoride decreased with addition of Al3+ by reducing of temperature,showed the participation of temperature-dependent transport.Na N3 also inhibited the accumulation of fluoride with addition of Al3+,possibly because of the transport of fluoride in different forms involved in energy-dependent transporter.The transport of fluoride also declined with addition of 2.5mg/L and 40 mg/L Al3+by Na N3,while 10 mg/L Al3+had minimum effect,indicated the limited effect of ATP in transport of Al F3,for the fluoride in the solution is mainly Al F3 with addition of 10 mg/L Al3+.Opening of tight junction with EDTA facilitated the transport of fluoride with addition of various concentration of Al3+,indicated the involving of paracellular transport in transport of fluoride.The transport of fluoride took place to a greater extent with addition of 10 mg/L Al3+,revealed the fact that Al F3 transport by paracellular pathway more easily.