An integrated strategy for rapid discovery and identification of the potential effective fragments of polysaccharides from Saposhnikoviae Radix

Saposhnikoviae Radix (SR) is a traditional Chinese medicine, known as “Fangfeng”. As one of the main active components, Saposhnikoviae Radix polysaccharides (SP) has demonstrated a range of biological activities, especially immunity regulation activity. This study aimed at exploring whether polysaccharides have activity after degradation, then discovering the potential effective fragments of SP. Here we establish the chromatographic fingerprints method for 32 batches of 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatives of oligosaccharides by HPLC, meanwhile evaluating its immunomodulatory activity in vivo. Then, the potential effective fragments of SP were screened out based on the spectrum-effect relationship analysis between fingerprints and the pharmacological results. Besides, liquid chromatography ion trap-time of flight mass spectrometry (LC-IT-TOF MS) coupled with multiple data-mining techniques was used to identify the potential effective oligosaccharides. These findings showed that the hydrolysate of SP have significant immunomodulatory, and the immunity regulation activity varies under different hydrolysis conditions. The 4 potential effective peaks of the hydrolysate of SP were mined by spectrum-effect relationship. Finally, the chemical structure of 4 potential effective oligosaccharide fragments of SP was elucidated based on LC-IT-TOF MS. F10 was inferred tentatively to be Hex1→6Hex1→6Hex1→6Hex1→6Hex1→6Gal; F18 was confirmed to be Rhamnose; F14 was inferred tentatively to be Hex1→4Hex1→ 4Hex1→4Gal and F25 was tentatively inferred to be Ara1→6Gal. This study may provide a sound experimental foundation in the exploration of the active fragments from macromolecular components with relatively complex structures such as polysaccharides.