Properties of fresh native A barbadensis glucomannan polysaccharide

Our discovery of the biological activity of the plant oligosaccharides arose during a systematic study of multiple biological activities in crude A. barbadensis extracts (Waller etal, 1994). This ARF Process A gel material was filleted with considerable attention to sanitation from prime leaves, depulped by passage though a 250 ^m commercial citrus depulper, and lyophilized (Waller etal, 1994; Pelley etal, 1998). Physico-chemically the polysaccharide in this Process A material was of a very high molecular weight.

Figure 12.4 Molecular weight distribution of polysaccharide isolated from Process A A. barbadensis gel as determined by gel filtration on Sepharose 4B.

Modified from Sheet 5, Figure 4C of Strickland etal. (1998). 10 g of Process A gel is suspended in 200 ml of water and dialysed across Spectraphor #1 tubing (MW cutoff 6—8 kDa) versus 4 liters water at 4 °C for 24 hours. This process is repeated four times until the conductivity of the retained material is <100 mSiemens. The desalted polysaccharide solution is then clarified by centrifugation for ~20kG for at least 15 minutes and the supernatant is further purified by precipitation at 80% v/v ethanol in the cold. Yields of polysaccharide from gel are in the range of 9 to 12% of solids. This purified polysaccharide was analysed by applying 30 mg in a volume of 3 ml to a 2.5 X40 cm column of Sepharose 4B and eluting with 0.0125% sodium azide at a flow rate of ~10 ml/hour. The eluate is analysed by the Dubois etal. (1956) phenol sulfuric acid method for hexose and the results expressed as absorbance at 490 nm. The three arrows represent respectively, the Vo of the column wherein molecules with MW > 2,000 kDa are excluded from the beads, the elution volume of a marker with a 90 kDa molecular weight and the Vt or total volume of the column. Three pools of eluted material, indicated by the dashed lines, are lyophilized and their hexose content determined. This profile is representative of three runs, each performed with polysaccharide isolated from a different Process A ARF Standard Sample. For more detailed information on this method consult the companion chapter by Waller et al. in this volume.

Analysis by Sepharose 4B gel filtration of the isolated polysaccharide gives a single peak with a long and variable tail (Figure 12.4). At least 60 ± 3% of polysaccharide has a MW of 2,000,000kDa or above and 83 ± 3% has a MW above 80kDa. However, up to 50% of the polysaccharide applied to the top of the column did not elute from the column, presumably because it was too large to easily flow through the bed of beads. However, it could be recovered by removing the top several cm of beads and washing the polysaccharide from them. Thus more than 90% of our ARF Process A gel polysac-charide is of a molecular weight at least ten-fold higher than anything previously described in the literature, excepting the allusion to insolublity in Gowda etal. (1979), referred to above. By comparison with the findings of McAnalley in Table 12.9, 10% of ARF Process A gel has a MW of 80kDA or lower, whereas 53% of experimental Acemannan® and 72% of commercial Acemannan® has a molecular weight of 80 kDa or lower.

On the other hand, the sugar analysis of the polysaccharide isolated from Process A gel (Pelley etal, 1998) gives a composition (85 ± 1% mannose, 7 ± 1% mannose and

Table 12.12 Role of time and cellulase dose in the activation and decay of the CHS response protective activity of A. barbadensis ARF Process A gel.

Time after Percent protection of CHS from suppression by 2000 Joules/m2

manufacture UV radiation (number of mice in group)

Concentration of cellulase (g. per 215 liters)

Time after Percent protection of CHS from suppression by 2000 Joules/m2

manufacture UV radiation (number of mice in group)

Concentration of cellulase (g. per 215 liters)

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