Study On The Absorption Mechanism Of Fucoidan In Vitro And Its Distribution In Vivo

Fucoidan is a class of fucose-rich sulfated water-soluble polysaccharide extraction from brown seaweeds.It exhibits a series of biological activities including anti-inflammatory,anti-tumor,anti-oxidant,reducing blood fat,antithrombotic,anti-viral and immunoregulatory.Due to the lack of a theoretical basis for the oral absorption mechanism of fucoidan and its tissue distribution in vivo,it is still difficult to develop it as a marine innovative drug that can be administered orally.At present,two key problems that hinder the oral absorption mechanism of fucoidan and its tissue distribution in vivo are as follows:First,due to the relatively high molecular weight of fucoidan,oral administration is difficult to be absorbed through biofilm.Secondly,the complexity of the structure of fucoidan makes the detection of polysaccharides in biological samples difficult.In this paper.FITC was used to label the fucoidan with different molecular weight,and then the absorption mechanism of the fucoidan was studied by a 7-day rapid culture model of Caco-2 cell in the transwell chamber.The tissue distribution of fucoidan in mice was also researched preliminary.The main parts of this dissertation are as follows:1)FITC labeled high and low molecular weight fucoidan.FITC was used to label high and low molecular weight fucoidan(HMWF and LMWF)respectively.The content of fucose and sulfate were measured before and after labeling.The content of fucose and sulfate in LMWF and FITC-LMWF were 62.86%and 67.11%,25.37%and 25.33%respective.The content of fucose and sulfate in HMWF and FITC-HMWF were 38.44%and 41.56%.19.54%and 20.97%respective,which showed no difference before and after tagged.We concluded that the method of FITC labelling fucoidan has no effect on the content of fucose and sulfate groups.The FITC-HMUT and FITC-LMWT exhibiting different green fluorescence bands by agarose gel electrophoresis,indicated that the HMWF and LMWF were labeled successfully.(2)The clathrin endocytosis pathway involved in the oral absorption mechanism of fucoidan.A 7-day absorption model of Caco-2 cell could be established quickly by adjusting the density of Caco-2 cells to 30×104 cells/cm2 and adding 0.4?g/mL puromycin without Penicillin-Streptomycin Solution.The TEER value of monolayer cells was higher than 500 ?*cm2 and the apparent permeability coefficient of fluorescent yellow transmittance(Papp)of monolayer membrane was less than 0.5 ×10-6 cm/s,which proved that the 7-day model could be used for oral drug absorption study in vitro.In this model,we found that the FITC-HMWF and FITC-LMWF can be absorbed with a higher Papp and absorptivity of FITC-LMWF than that of FITC-HMWF.There was also a negative correlation between the concentration and the Papp of the both fucoidans,which suggested that the absorption of fiucoidan may depend on carrier.The result of blocking clathrin endocytosis pathway by inhibitors(CPZ,NH4CL,Dynasore)demonstrated that there was significant reduction in the Papp and absorptivity of FITC-LMWF respective.The inhibition ratio declined by 84.24%,74.61%,63.94%respectively.We concluded that the clathrin endocytosis pathway may participate in the oral absorption of fucoidan.(3)The distribution of fucoidan in vivoWe verified that the free FITC did not interfere with the detection of FITC-LMWF in tissue samples and the FITC-LMWF at a dose of 2500 mg/kg injected to mice did not cause toxicity.The tissue distribution of FITC-LMWF after intravenous injection administration at the dose of 50 mg/kg to the mice was characterized,and it exhibited considerable heterogeneity.Fucoidan can quickly distributed into kidney and liver and preferentially accumulates in the kidneys with the content of 1092.31 ?g/g.284.27 ?g/g respective higher than other organs.We can speculat that kidney and liver might be the targeted organs of fucoidan.In conclusion.HMWT and LMWF can be labeled by FITC successfully,and 7-day model of Caco-2 cell is used to verify that clathrin endocytosis pathway is involved in the absorption and transport of fucoidan.The fucoidan can rapidly distribute into tissues and accumulate into the kidney and liver preferentially,so it is presumed that the kidney and liver may be its target organs.All results suggest that HMWF and LMWF can be absorbed orally,and the LMWF absorptivity is higher,and the liver and kidney may be the target organs of fucoidan,which provides a theoretical basis for the further development of liver and kidney drugs.