Preliminary Study On The Intestinal Absorption And Pharmacokinetics Of Fucoidan From Laminaria Japonica

Fucoidans are a class of fucose-rich sulfated water-soluble polysaccharide extraction from brown seaweeds and echinoserms.It has been reported that fucoidans exhibit many biological activities,such as anti-cancer,immune modulation,anti-pathogen,anti-inflammatory,anti-thrombotic,anti-oxidant,treatment for liver and kidney health.Although many studies have reported its biological activities,few have examined the pharmacokinetics of fucoidan after intragastric administration.Generally,polysaccharides with high molecular weight are not considered to be absorbed orally,however,it's reported that Undaria pinnatifida fucoidan,Cladosiphon okamuranus fucoidan and tea plant flower polysaccharide can be absorbed by oral administration.China has rich resources of Laminaria japonica.The study on the absorption of fucoidan from Laminaria japonica is not only beneficial to the comprehensive utilization and deep development of the Laminaria japonica resource,but also is helpful for the development of fucoidan into medicine.In this study,a novel fucoidan content determination method was established by FITC fluorescent labeling.The absorption of fucoidan and its mechanism were investigated by single-pass intestinal perfusion technique for in situ intestinal absorption and Caco-2 cell model.Besides,the pharmacokinetic characteristics of fucoidan were studied by oral administration.The experimental process and results are as follows:1.Fluorescent labeled fucoidan can improve the sensitivity and specificity of the content determination method.The FITC was used to label fucoidan with fluorescence properties and the sensitivity and specificity of the content determination method was improved.The result of the labeled experiment was verified by fluorescence spectrophotometer and high performance liquid chromatography.On the basis of fluorescence labeling,the method of measuring the content of fucoidan by external standard method and internal standard method was established by using high performance liquid chromatograph with fluorescence detector,and the validation of this method was studied.The results showed that the external standard method and internal standard method are in accordance with the provisions of the Chinese Pharmacopoeia.The lower limit of detection of the external standard method and internal standard method were 0.05?g/mL and 0.5 ?g/mL,respectively.It can meet the test sensitivity required for the experiment.2.Fucoidan can be absorbed by the intestineApproximately 10 cm long duodenum,jejunum,and ileum were partially cut opened at both ends respectively.The concentrations of fucoidan were 0.01 mg/mL,0.1 mg/mL and 1 mg/mL as low,medium and high perfusion concentration,respectively.The results showed that fucoidan was the best absorption in the jejunum,followed by ileum and the duodenum,indicating that the main absorption parts for fucoidan were the jejunum and ileum.In addition,the absorption at low concentrations is better.The high concentration of fucoidan was inhibited,indicating that the absorption of fucoidan may be dependent on the carrier transport.3.Fucoidan can be absorbed at the Caco-2 cell model and the absorption mechanism is endocytosisThe Caco-2 cell model was established and the tightness of the cell model was detected by TEER resistance meter.The Caco-2 cell model was used to examine the uptake and efflux of fucoidan in intestinal cells.The results showed that the absorbed amount of the fucoidan in the model was few,but the absorption value was observed and the absorption was greater than the efflux.It was indicated that fucoidan can enter the blood circulation through the intestinal.In addition,the effects of temperature,clathrin inhibitor,concentration and time on its absorption were investigated by Caco-2 cell model,respectively.For the effect of transport time on absorption of fucoidan,30,60,90,120,180,240 min was selected as the point of time for the studying of absorption.The results showed that the Papp value reached the maximum at 120 min,and then the absorption process gradually became stable.For the effect of the concentrations of sample on absorption of fucoidan,the concentrations of fucoidan were 0.01 mg/mL,0.1 mg/mL and 1 mg/mL as low,medium and high concentration,respectively.The results showed that the absorption rate was significantly decreased at high concentration.It was further verified that the absorption of fucoidan was dependent on the transport of the carrier.For the effect of the temperatures on absorption of fucoidan,temperatures were selected at 4? and 37?,respectively.The results showed that fucoidan could be absorbed by intestinal tract at 37?,and almost no absorption at 4?.It was indicated that the absorption of fucoidan is a process of energy consumption.Besides,the chlorpromazine hydrochloride was added to investigate the impact of clathrin inhibitor for its absorption process.The experimental results showed that chlorpromazine hydrochloride group was almost no absorption,and it was speculated that fucoidan may be through the endocytosis for absorption.4.Pharmacokinetic characteristics of fucoidan was studied in ratFucoidan dissolved in ultra-pure water to be prepared as a 10 mg/mL sample solution.According to the weight of rats administered.The fucoidan was administered intravenous at a dose of 6 mg/kg and orally at a dose of 20 mg/kg to rats.0.3 mL of blood samples were serially withdrawn from the caudal vein into antigoagulant tubes with 0.45 mg EDTA at 0,0.5,1,2,3,4,6,12 and 24 h after oral administration and intravenous administraion.The plasma samples were pretreated by 10%trichloroacetic acid to precipitate protein and ethanol to precipitate polysaccharide.The Cmax was 7.33 ?g/mL within 2 h by orally administration.The bioavailability was 2.67%of the fucoidan by orally administration in rats.In summary,fucoidan from Laminaria japonica can be absorbed by the intestine.And the absorption at the Caco-2 cell model is greater than the efflux effect.The mechanism of its absorption may be endocytosis depending on the clathrin.Pharmacokinetic experiments show that fucoidan can enter into the blood circulation and the oral bioavailability was 2.67%.