Studies On Labeling Of Grifolan With Fluorescein Isothiocyanate And The Absorption And Transport Of It In Caco-2 Cells Model

The polysaccharide of Grifola frondosa(Grifolan, GRN) has many kinds of biological activity, mainly including anti-tumor, strengthen the body’s immune especially the immune cells. In the clinical, GRN often used in combination with chemotherapy drugs. GRN not only can improve the pharmacodynamic but also can reduce the side effects of drugs. However, there is no one has to study the pharmacokinetics of the polysaccharide of Grifola frondosa, the main difficulty lies in the lack of high sensitive maitake polysaccharide detection technology. The main difficulty is that it’s lack of high sensitive detection technology of the polysaccharide of Grifola frondosa. We aimed to use FITC to label GRN and through Caco-2 cell model to study the absorption and uptake for GRN. Promoting the further pharmacokinetic study of GRN.First of all, use Sevag, fractional precipitation and any other methods to achieve the effect of preliminary purification of GRN, The fine purification GRN was obtained after chromatography on DEAE-cellulose and Sephadex G-100 column. By using phenol-sulfuric acid method determinated the total polysaccharides content of GRN was 95.7%.The second, the reducing terminal of GRN was used to selectively insert tyramine(Tyr) by covalent coupling. Through the membrane separation technology to remove Tyr and other impurities. Then labeling the GRN-Tyr with fluorescein isothiocyanate(FITC) by nucleophilic reaction. The pure GRN-Tyr-FITC was obtained after chromatography on Sephadex G-100 column. By using phenol-sulfuric acid method determinated the glycogen content in each step of reaction products. Comparing the electrophoresis mobility(Rx) of each reaction products by PAGE. Through UV spectrum scanning the products if there is a characteristic absorption peak in 280 nm, 490 nm. Through fluorescence spectrum scanning GRN-Tyr-FITC if the optimal excitation wavelength(Ex) and emission wavelength(Em) are changed. Calculate the degree of substitution of FITC in GRN-Tyr-FITC and carry out the stability testing. The results show that GRN-Tyr and GRN-Tyr-FITC marked success. Under the condition of Ex: 490 nm, Em: 520 nm, GRN-Tyr-FITC can be used for fluorescent quantitative detection experiments. Although there is a slight fluorescent red shif in the Ex and Em of GRN-Tyr-FITC. Under the condition of 4 °C and avoiding light, GRN-Tyr-FITC can stable preservation for 30 days. The degree of substitution of FITC in the range of 0.039%~0.060%(W/W) in batch type synthesis of GRN-Tyr-FITC.Finally, using a simple and rapid culture method of Caco-2 cells to simulated intestinal absorption and transport polysaccharide-Transwell absorption model. Establish Caco-2 cell monolayer culture model and use detection of TEER, microscopic morphology observation and markers seepage methods to detect the completeness and compactness. Then GRN are made of 100μg/mL, 200μg/mL, 400μg/mL, 600μg/mL, 800μg/mL and 1000μg/mL. Add to the Caco-2 cell monolayer in Transwell absorption model. Set 30 min, 60 min, 120 min, 180 min, 240 min for testing point and evaluation the transport ability of Caco-2 cells in the the direction of the AP-BL and BL-AP. After the test, Testing TEER again and carry out the recovery experiment. The results show that there is probably active transport mechanism of GRN-Tyr-FITC in AP. The uptake of Caco-2 cells for GRN-Tyr-FITC haven’t reach saturation in 0-4h. But the uptake of Caco-2 cells for GRN-Tyr-FITC(600-1000μg/mL) have reach saturation in BL after 180 minutes. TEER does not change significantly compared with previous experiments. The recovery rate of GRN-Tyr-FITC was 64.52%(W/W). The PAGE electrophoresis electrophoresis mobility is similar between the recovery liquid of GRN-Tyr-FITC and the control group of GRN-Tyr-FITC. This suggests that the uptake of Caco-2 cells for GRN-Tyr-FITC did not affect its structure. Bes ides, the fluorescence labeling method is suitable for the pharmacokinetic analysis of this kind of GRN.