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

Bin Yang; Yuan Li; Wentao Shi; Yingying Liu; Yongjun Kan; Jinlong Chen; Juan Hu; Wensheng Pang

doi:10.3390/molecules27103192

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

Molecules. 2022 May 17;27(10):3192. doi: 10.3390/molecules27103192.

Authors

Bin Yang^{1

2}, Yuan Li^{1

2}, Wentao Shi^{1

2}, Yingying Liu¹, Yongjun Kan³, Jinlong Chen², Juan Hu^{1

2

3}, Wensheng Pang²

Affiliations

¹ The Second Affiliated Hospital of Fujian University of Traditional Chinese Medicine, Fuzhou 350003, China.
² School of Pharmacy, Fujian University of Traditional Chinese Medicine, Fuzhou 350122, China.
³ Institute of Materia, Fujian Academy of Traditional Chinese Medicine, Fuzhou 350003, China.

Abstract

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, PHP_0.5MSC-F and PHP_H-1-2, which have anti-hyperglycemia and insulin resistance improvement effects for type 2 diabetes. In this study, the reducing terminal sugars of PHP_0.5MSC-F and PHP_H-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 PHP_0.5MSC-F was found to be 8.84 μg/mL with a linear range of 29.45-683.28 μg/mL. In reference to T70, the detection limit for PHP_H-1-2 was found to be 13.89 μg/mL with a linear range of 46.29-462.76 μg/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 × 10^-6 cm/s, which suggests easy absorption. Regarding bidirectional transport, the AP→BL Papp values were greater than the BL→AP values; thus, PHP_0.5MSC-F and PHP_H-1-2 mainly underwent passive transference. The two membrane permeable polysaccharides were not P-gp efflux transporter substrates. The absorption mechanism of PHP_0.5MSC-F complies with passive diffusion under a concentration gradient, whereas PHP_H-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.

Keywords: 2-AB fluorescent labeling; HPLC-FLD; Pseudostellaria heterophylla polysaccharides; bi-directional transport; caco-2 cell.

MeSH terms

Biological Transport
Caco-2 Cells
Diabetes Mellitus, Type 2*
Humans
Polysaccharides
Tissue Distribution

Substances

Polysaccharides

Grants and funding

The authors give thanks to Project 81872994, supported by the National Natural Science Foundation of China; the Fujian Provincial Science and Technology Plan for foreign cooperation projects (2019I0015); the Central Committee to Guide Local Science and Technology Development (Grants No. 2020L3012); and Project 2019YFC1710504, supported by the National Key R & D Plans.