Extraction, Isolation, Purification And Structural Identification Of Polysaccharides From Humulus Lupulus

Humulus lupulus is one of the basic ingredients for brewing beer,it can not only give the beer unique aroma and fresh bitterness,but also improve the clarity and stability of the beer.Studies have proved that H.lupulus have many kinds of biological activities,but the bioactive ingredients responsible for its beneficial effects remain unclear.Our research group pre-purified and identified two homogenous H.lupulus polysaccharides,HL70-2 and HL90-1,and found that they have good osteogenic activity in vitro.Therefore,this paper continued to study the separation,purification and structural characterization of H.lupulus polysaccharides,which has provided a basis for the subsequent researches on the mechanism of anti-osteoporosis and the structure-activity relationship of polysaccharides from H.lupulus.In this study,hot water and alkaline solution were used to extract the polysaccharides from female flowers of H.lupulus.The crude polysaccharides HL50,HL70,HL90,and HLB were obtained by fractional alcohol precipitation,and the sugar contents measured by the phenol-sulfuric acid colorimetric method were 52.3%,79.8%,49.7%,and 72.3%,respectively.The crude polysaccharides HL50,HL70,and HLB were deproteinated by the Sevage method,dialyzed against distilled water,concentrated,freeze-dried,and subjected to separation with DEAE-52 cellulose and Sephadex G-75 gel filtration chromatography to obtain eight kinds of polysaccharides,HLP50-1,HLP50-2,HLP70-2-1,HLP70-2-2,HLP70-3,HLBP-2,HLBP-3,and HLBP-4.The specific optical rotation and high-performance gel permeation chromatography verified that the above eight polysaccharides were homogeneous polysaccharides with molecular weights of 49.13 kDa,73.25 kDa,11.12 kDa,7.3 kDa,26.48 kDa,6.13 kDa,35.23 kDa,and 33.12 kDa,respectively.Monosaccharide composition analysis used PMP derivatization,combined with the D/L absolute configuration analysis,infrared analysis,full methylation-GC-MS analysis,and nuclear magnetic resonance analysis to obtain the primary structural characteristics of these eight homogeneous polysaccharides.The results proved that these eight homogeneous polysaccharides from H.lupulus were heteropolysaccharides formed from multiple sugar residues,and the structure has not been reported.Congo-red and scanning electron microscopy experiments were used to conduct preliminary studies on the advanced structures of polysaccharides.Congo-red experiments showed that the eight homogeneous polysaccharides did not contain triple-helix structure.Scanning electron microscopy results proved that the surface topography of the eight homogeneous polysaccharides were different.