Isolation, purification, structural analysis and coagulatory activity of water-soluble polysaccharides from Ligustrum lucidum Ait flowers

Abstract

In this study, Ligustrum lucidum flowers as raw material, the extraction, isolation and coagulatory activity of polysaccharides were carried out for the first time. The crude polysaccharide was obtained by hot water extraction and ethanol precipitation, and preliminarily purified by Sevage method and D101 macroporous resin. Then the polysaccharide was further purified by DEAE-52 cellulose and Sephadex G-100 column chromatography, respectively. The structural characteristics were detected by LC, GC, FT-IR and NMR. Furthermore, the coagulatory activity of the polysaccharides were investigated by APTT, TT, PT and FIB assays in vitro. The results demonstrated that four polysaccharides were isolated from flowers of L. lucidum, named as LLP-1a, LLP-1b, LLP-2 and LLP-3, and the yields were 0.039, 0.0054, 0.0055 and 0.017%, respectively based on the weight of the dried flowers. The four polysaccharides components were free of nucleic acids and proteins, and their average molecular weights were 25,912, 64,919, 3,940,246 and 2,975,091 g/mol, respectively. The monosaccharide compositions of LLp-1a were l-rhamnose, l-arabinose, d-xylose, d-glucose and d-galactose (molar ratio of 3.16: 2.46: 1.00: 7.27: 4.22). Only d-galactose was detected from LLp-1b. LLp-2 was composed of l-arabinose, d-glucose and d-galactose (molar ratio of 1.28:1.32:1.00). LLp-3 was composed of l-rhamnose, l-arabinose, d-xylose, d-glucose and d-galactose (molar ratio of 5.85: 2.21: 2.23: 1.00: 2.25). Coagulation assays indicated that LLp-1a and LLp-3 had good anticoagulant effect in vitro, while LLp-1b showed procoagulant activity.

Antioxidant Activities of an Exopolysaccharide (DeinoPol) Produced by the Extreme Radiation-Resistant Bacterium Deinococcus radiodurans

“…Production of GalactoPol and FucoPol isolated from Pseudomonas oleovorans and Enterobacter A47, respectively, are maximized under aerobic conditions, with carbon availability concomitant with nitrogen and oxygen limitation 38 . In contrast, gellan and cellulose production is favored in media supplemented with vitamins and amino acids [39][40][41][42][43][44] . We also investigated the optimal culture condition for DeinoPol production based on its sugar composition analyzed by Bio-LC mass spectrometry in this study.…”

“…We used gradient ethanol precipitation to purify DeinoPol, but its high toxicity (LD 50 = 12.5 μg/mL) presents a limitation for its use as a cosmetic ingredient. We, therefore, used the Sevage method after ethanol precipitation to remove toxic, insoluble materials such as lipids and membrane proteins [41][42][43] . The final product was shown to be non-toxic at over 1000 μg/mL in NHEK-Ad (Supplement ary Fig.…”

“…However, the purity of DeinoPol was likely to be less than 90% based on gel filtration chromatography and on protein and nucleic acid concentration analyses. To obtain a higher grade of DeinoPol, additional processes should be performed following precipitation and the Sevage method, such as protein removal by protein precipitation with trichloroacetic acid or proteinase treatment 42,43 .…”

“…DeinoPol was extracted, isolated, and purified as described in previous studies [39][40][41][42] with slight modifications. D. radiodurans R1 strain was cultured in 4 L of TGY broth at 30 °C, shaking at 900 rpm.…”

“…The concentrated supernatant (approximately 40 mL) was precipitated with 160 mL of 95% ethanol (Daejungchem; Seoul, Korea) at 4 °C for 12 h, and the precipitate was collected by centrifugation at 5000 × g for 10 min at 4 °C to yield the crude polysaccharide solution. The proteins in the crude polysaccharide were removed by the Sevage method as described previously 41,42 . Briefly, the precipitate was dissolved in 10 mL of distilled water and mixed with 30 mL chloroform:n-butanol (4:1 v/v).…”