Use of Fluorescent 2-AB to Explore the Bidirectional Transport Mechanism of Pseudostellaria heterophylla Polysaccharides across Caco-2 Cells

Polysaccharides are abundant in natural resources and perform numerous physiological functions. Polysaccharide structures often lack chromophore groups; thus, current analytical methods cannot distinguish polysaccharide metabolites in the body or polysaccharide prototypes in biological samples. Thus, the measurement of polysaccharides in blood, bodily fluid, and cell-culture medium is difficult. Our early-stage research resulted in the isolation of two homogeneous polysaccharides from Pseudostellaria heterophylla, PHP0.5MSC-F and PHPH-1-2, which have anti-hyperglycemia and insulin resistance improvement effects for type 2 diabetes. In this study, the reducing terminal sugars of PHP0.5MSC-F and PHPH-1-2 were labeled with 2-aminobenzamide (2-AB) to prepare novel fluorescent probes for HPLC-coupled fluorescence detection (HPLC-FLD). Quantitative analysis was performed in reference to T40, and the detection limit for PHP0.5MSC-F was found to be 8.84 mu g/mL with a linear range of 29.45-683.28 mu g/mL. In reference to T70, the detection limit for PHPH-1-2 was found to be 13.89 mu g/mL with a linear range of 46.29-462.76 mu g/mL. This method was used to measure the bidirectional transport of polysaccharides across caco-2 cells from apical to basolateral (AP -> BL) or from basolateral to apical (BL -> AP) directions and to evaluate the polysaccharide bioavailability. The drug absorption capacity was determined based on the apparent permeability coefficient (Papp), and the Papp values for the two polysaccharides were found to be greater than 1 x 10(-6) cm/s, which suggests easy absorption. Regarding bidirectional transport, the AP -> BL Papp values were greater than the BL -> AP values; thus, PHP0.5MSC-F and PHPH-1-2 mainly underwent passive transference. The two membrane permeable polysaccharides were not P-gp efflux transporter substrates. The absorption mechanism of PHP0.5MSC-F complies with passive diffusion under a concentration gradient, whereas PHPH-1-2 mainly utilizes a clathrin-mediated endocytic pathway to enter caco-2 cells. This innovative HPLC-FLD method can help to track polysaccharide internalization in vitro and in vivo to facilitate cellular uptake and biodistribution exploration.

Automated summary

Polysaccharides, which are abundant in natural resources and have various physiological functions, are difficult to measure in biological samples due to the lack of chromophore groups. In this study, two homogeneous polysaccharides from Pseudostellaria heterophylla were isolated and labeled with a fluorescent probe for detection. The developed method enabled the tracking of polysaccharide internalization and biodistribution exploration.