An ethanol extract of aloes was found to have strongly hemagglutinating effects (Bouthet etal, 1996). Lectins or lectin-like substances had been generally prepared as water-soluble substances. However, an ethanol extract of A. barbadensis was described to have lectin-like properties, i.e. strong hemagglutinating effects toward human erythrocytes. In this study, the recognition sites of specific sugars with ethanol extract was particularly discussed and identified.
A commercially available gel filet of A. barbadensis leaves in irradiated, lyophilized form was reconstituted into a solution with double distilled water. The aloe extract was obtained by two subsequent ethanol precipitations at 50% in absolute ethanol. The pellet after centrifugation was resuspended in water and dialyzed against water. Then the extract was lyophilized and reconstituted into solution with water.
Hemagglutination activity of the aloe extract was tested toward human erythrocytes. The extract was originally dissolved in water and phosphate-buffered saline (PBS) was used for further dilution to avoid osmotic shock to the erythrocytes, because it was more soluble in water than in the other buffers.
The hemagglutination of the aloe extract was not blood type dependent and 13 monosaccharide sugars (fructose, galactosamine, galactose, N-acetyl galactosamine, D-fucose, glucosamine, N-acetyl glucosamine, mannose, mannosamine, O-methyl-mannose, muramic acid, N-acetyl mannosamine and L-rhamnose) and four disaccharides (chondrosine, maltose, raffinose and sucrose) were examined for their hemagglutination inhibition. Glucosamine, mannosamine and chondrosine inhibited hemagglutination induced by the aloe extract. The inhibition activity was strongest with glucosamine and weakest with chondrosine.
Factors influencing hemagglutination. ii pH, temperature, trypsin, EDTA
The aloe extract was heat and trypsin resistant (but using bovine pancreatic type I protease) and pH sensitive. Acid treatment (pH 3) of the aloe extract caused a decrease of the hemagglutination activity, whereas high pH (12) treatment did not change the activity. The addition of EDTA to hemagglutination assay caused a decrease of the hemagglutination activity, indicating that Ca2+ is required for the hemagglutination.
SDS-PAGE showed 12 polypeptides of the aloe extract with the following approximate molecular masses: 83, 81, 75, 67, 63, 51, 47, 38, 28, 21, 15 and 12kDa. Two bands with molecular mass of 15 kDa and 12 kDa seemed to be major components of the aloe extract.
Recognition sites of the specific sugar to the aloe extract
The a-amino at the C2 position and a-hydroxyl at the C4 position of glucosamine were concluded to be critical for binding to the aloe extract. The conclusion was introduced by comparisons of the chemical structures of mannosamine, muramic acid, galactosamine and chondrosine.
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