Objective  To study the standard extract of hedysarum polysaccharide (HPS), and to screen and optimize its "deproteinization-macroporous adsorption resin purification and enrichment" process method and process parameters.

Methods  Taking the protein removal rate and polysaccharide loss rate as indexes, the deproteinization effects of four different methods were compared, and the process parameters of the selected methods were optimized. Taking adsorption capacity and adsorption rate as indicators, the purification and enrichment effects of four types of macroporous adsorption resins (AB-8, D-101, NKA-9, X-5) on HPS were investigated, and the related process parameters were optimized. Phenol sulfuric acid method and Coomassie brilliant blue method were used to determine the content of polysaccharide and protein. The basic characteristics of polysaccharides before and after purification were compared by UV and IR methods.

Results  Sevag method was selected for deproteinization, and AB-8 macroporous adsorption resin was the preferred resin. Through the optimization of process parameters, the purification process was determined as follows: 10 mg·mL^-1 crude polysaccharide aqueous solution, deproteinization twice with 1/2 volume of chloroform-n-butanol (4 ∶ 1, v/v) mixed reagent; after the residual organic solvent was removed, the pretreated AB-8 macroporous adsorption resin column was loaded with 1.5 mg·mL^-1 aqueous solution [the ratio of resin amount (g) to loading volume (v) was 1 ∶ 10], and the sample was loaded twice at a flow rate of 2.0 mL·min^-1. After dynamic adsorption, static adsorption was carried out for 6-8 h, and then elution was carried out with 1.0 mL·min^-1 of 30% ethanol until the eluate had no obvious basic copper tartrate reaction, the eluate was collected and concentrated to 1 ∶ 1 with the original drug, anhydrous ethanol was added to 80% alcohol content, the precipitate was precipitated for 10 h, then filtrated it and the precipitate was rinsed with anhydrous ethanol and acetone and the HPS was extracted.

Conclusion  The verification experiment showed that the purity of HPS sample was increased by 1.27 times after purification and enrichment by this process. There was no protein absorption peak at 260 nm and 280 nm in UV spectrum. IR spectrum showed more prominent absorption peaks of polysaccharide structure characteristics, which indicated that the purification process was effective and had no influence on the main structure of HPS. The optimized HPS deproteinization and macroporous resin purification process is stable and feasible, with good repeatability, so that the polysaccharide can be purified and enriched, which provides a research foundation for the formation of HPS standard extract and its ability to become a scientific raw material of preparations.

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