On the isolation of immunostimulatory active acemannan from Aloe barbadensis
Abstract
Acemannan from Aloe barbadensis was obtained with four processes: 1) size exclusion chromatography (SEC) using Sepharose CL-4B matrix followed by ethanolic precipitation; 2) SEC, ultrafiltration using hollow-fiber cartridges (30 kDa or 0.1 µm) and ethanolic precipitation; 3) SEC, precipitation with cetyltrimethylammonium bromide (CTAB) and ethanolic precipitation; and 4) direct precipitation with CTAB and ethanolic precipitation. The detergent CTAB was effective to concentrate chromatographic eluates (process 3) and allowed the direct isolation and purification of acemannan from crude ethanolic extracts (process 4), without recurring to SEC. Process 4 also decreases operation time (9 days vs. 15 days in process 3), and costs regarding raw materials. Both processes generate materials devoid of detectable levels of anthraquinones and contaminating DNA, and proteins below 5% of dry weight. This material was essentially made of mannose; 97% obtained in processes 1-3 and 75% in process 4, with a molecular mass ranging from 2000 to 5000 Mr according to G5000 PW SEC. Acemannan in dry form was sterilized at the optimal 10 kGy ?-radiation dose, and retained both its physical-chemical properties and adjuvanticity for HBsAg co-delivered by the nasal route in mice. The mixture of acemannan-1% benzyl alcohol (w/v) does not affect the adjuvanticity. The total carbohydrates content, SEC-HPLC, pH, microbial limit and organoleptic characteristics of the irradiated polysaccharide suspended in phosphate buffer remained stable at -20 ºC for at least 6 months of storage. These results may be useful for designing processes for producing pharmaceutical quality acemannan to be used in vaccine clinical studies.
