Isolation, Purification And Structure Analysis Of The Polysaccharides From Apocynum Venetum Leaves And Its Interaction With Galectin-3

Apocynum venetum Linn (AV.) has been used as traditional medicine, in China. The leaves of AV. have many bioactivities, such as antiatheroscloresis, hepatoprotective effect, antidepressie and so on. Up to now, there were nearly 20 papers reported the studies on the leaves of AV., which were about polyphenols and glucosides. But no reports on isolation, purification, structure analysis and bioactivity of the polysaccharides from AV. leaves.In this paper, we reported the total fractionation of the water-soluble AV. leaves polysaccharides. The monosaccharide compositions, physico-chemical properties and molecular weight distributions of all fractions were analyzed and the structure features of the homogeneous fractions were studied in details.The crude water-soluble polysaccharides (ALP, 5.8%) of dry AV. leaves was extracted with water, precipitated with ethanol and deproteinated by Sevag method. The result of monosaccharide composition indicated that ALP contained Man: Rha: GlcA: GalA: Glc: Gal: Ara in a molar ratio of 0.29: 1.0: 0.55: 3.88: 1.12: 2.20: 1.29. ALP was loaded on DEAE-Cellulose column to obtain one neutral fraction (ALPN) and two acidic fractions (ALPA-1 and ALPA-2) . All fractions were analyzed by the contents of sugar, monosaccharide compositions, molecular weight distributions. ALPA-1 and ALPA-2 were further fractionated to homogeneous frations ALPA-1a and ALPA-2a.The results showed that ALPA-1a was homogenous with molecular weight of 5.0×104 Da. High performance liquid chromatography (HPLC) indicated that ALPA-1a was mainly consisted of Rha (9.93%) , GalA (8.2%) , Gal (37.55%) and Ara (36.92%) . The amount of Gal and Ara acounted for over 50% of total sugars. The ratio of Rha/GalA and Gal/Ara were 1: 1, respectively. 13C NMR spectroscopy showed that ALPA-1a possessed a backbone ofα-D-GalpA- (1, 2) -α-L-Rhap with the side chains of arabinogalactan residues. All the results indicated that it belongs to type-I rhamnogalacturonan (RG-I) -rich pectin.ALPA-2a was homogenous with molecular weight of 1.8×104 Da. High performance liquid chromatography (HPLC) indicated that ALPA-2a was mainly consisted of GalA, which approached to 70%. Furthermore, it also contained Rha (7.38%) , Glc (8.29%) , Gal (7.07%) and Ara (5.43%) . The structural features of ALPA-2a were elucidated by a combination of IR spectra, periodate oxidation, partial acid hydrolysis and 13C NMR spectroscopy. GalA was in the form ofα-D- (1, 4) , which made up the backbone in ALPA-2a, with someα- (1, 2) -Rhap residues. The side-chains also contained arabino- (3, 6) -galactan domain. All the results indicated that it belongs to homogalacturonan (HG) -rich pectin. In the hemagglutination inhibition assay, it is revealed that ALPA-1b and ALPA-2a could both inhibit hemagglutination on lower concentration than positive control, namely, which had very strong binding with Galectin-3. Furthermore, ALPA-1a also has strong binding with Galectin-3, and that ALPN has no effect on Galectin-3. According to the structure characteristic of all the fractionations, we presumed that HG-rich pectin probablely had stronger binding with Galectin-3 than RG-I-rich pectin. But AG-rich pectin almost did not bind with Galectin-3, which maybe associate with the special structure characteristic of HG-rich pectin or the linkage forms and spatial positions of galacose in ALPA-1b and ALPA-2a.