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Volume of Elute

Figure 12.6 Crude Process A A. barbadensis gel was incubated with 0.5 g per 215 liters cellulase, the polysaccharide isolated by dialysis /ethanol precipitation and molecular weight distribution determined by gel filtration on Sepharose 4B.

Modified from Sheet 5, Figure 4D of Strickland etal. (1998). This material was from the same lot of gel as illustrated in Figure 4 except that it was incubated with 'cellulase'. Subsequent to incubation, the polysaccharide was isolated and analysed by the same method as in Figure 4.

of action. A second factor can be removed by common commercial processing steps. There may even be a third factor, distinct from either of the two above.

Studies on cellulase cleavage of purified native polysaccharide

Investigations to determine the nature of the skin immune system (SIS) UVB-protective and macrophage stimulating molecules revealed a polysaccharide was purified from Process A gel under conditions (see flow chart Figure 5, sheet 7 in Strickland etal, 1998) which resulted in minimal alteration of the gel as demonstrated by the polysaccharide molecular weight distribution analysis in Figure 12.7A below.

The purified polysaccharide was then incubated with various concentrations of partially purified T. reesei 'cellulase' and the reaction terminated by alcohol addition, separating the alcohol-soluble oligosaccharides (see flow chart Figure 6, sheet 8 in Strickland etal, 1998; Byeon etal, 1998; Strickland, 1999). This treatment progressively reduced psuedoplasticity (Figure 12.8) and altered the molecular weight distribution profile of the polysaccharide. Instead of observing only the ultra-high molecular weight form of the native polysaccharide, some material appeared that resembled Acemannan® (Figure 12.7B, region eluting between 160 to 200ml). All the applied polysaccharide was able to migrate through the column. However, the degree of breakdown of polysaccharide was not as extreme as that which occurs in industrial type processing with even the lowest dosage of 'cellulase' (compare Figure 12.7B to Figure 12.6). This observation, that pseudoplasticity is 'broken' and biological activity is generated more readily than Acemannan® structure is digested, has prompted us to propose that there is a highly 'cellulase' sensitive site near the cross-links in the native polysaccharide gel (Figure 12.3).

Studies by Dr Sheffield indicated that the ethanol-insoluble polysaccharide fraction cleaved from native polysaccharide by 1 ^g, 3 ^g and 10 ^g 'cellulase' per 100 mg

o 100 200 o 100 200 300

Volume of Bute Volume of Bute o 100 200 o 100 200 300

Volume of Bute Volume of Bute

Figure 12.7 Molecular weight distribution of, A. Polysaccharide purified from Process A A. barbadensis gel and incubated in buffer and B. Purified polysaccharide incubated for 2.5 hours with 30 |lg cellulase per 100 mg polysaccharide.

Modified from Figure 8, Sheets 10 and 11 of Strickland et al. (1998) and conducted according to the protocol of Figure 6, Sheet 8. The chromatographs are representative of two runs each, conducted with materials from different preparations. After incubation and prior to chroma-tography, the polysaccharide was concentrated by precipitation with ethanol.

Figure 12.7 Molecular weight distribution of, A. Polysaccharide purified from Process A A. barbadensis gel and incubated in buffer and B. Purified polysaccharide incubated for 2.5 hours with 30 |lg cellulase per 100 mg polysaccharide.

Modified from Figure 8, Sheets 10 and 11 of Strickland et al. (1998) and conducted according to the protocol of Figure 6, Sheet 8. The chromatographs are representative of two runs each, conducted with materials from different preparations. After incubation and prior to chroma-tography, the polysaccharide was concentrated by precipitation with ethanol.

0 50 100 150

Time (minutes)

Figure 12.8 Change in relative viscosity of polysaccharide purified from Process A A. barbadensis gel and incubated with T. reesei cellulase.

Modified from Figure 7, sheet 9 of Strickland et al. (1998) and conducted according to the protocol of Figure 6, Sheet 8 using 100 mg portions of polysaccharide.

polysaccharide, contained the phagocyte activating activity (Strickland etal, 1998, Figure 9). The ethanol soluble oligosaccharide fraction contained the CHS protective activity (Strickland etal, 1998).

Native polysaccharide incubated in the absence of cellulase failed to generate activity protective of CHS from suppression by UVB (Table 12.13). Protection was achieved by

Table 12.13 CHS-UVB protective activity of oligosaccharides cleaved from

A. barbadensis polysaccharide.

Oligosaccharide concentration

Percent protection of CHS from suppression by 2000 Joules/m UV radiation

Cellulase/polysaccharide concentration

None

Note

Values summarize data from Table 7 in Strickland etal. (1998). Percent protection is calculated according to Formula 2. Statistical significance of protection, 3 ^g cellulase,

oligosaccharides generated by moderate (3 ^g/100mg) ' cellulase' cleavage of native polysaccharide, albeit this factor demonstrated significant suppression at supraoptimal doses. Cleavage of polysaccharide with excessive amounts of cellulase (30 ^g/100mg) resulted in inactive preparations. A similar SIS protective activity was observed with assays utilizing DTH (Byeon etal, 1998, Table I).

Other parallel studies by Dr Sheffield led us to name this novel oligosaccharide activity the cytoprotective oligosaccharide (L. Sheffield, unpublished observations). It was first noted that crude extracts of A. barbadensis gel accelerated heat shock recovery, that is the re-entry of cultured cells into the cell cycle after thermal injury. This is measured as DNA synthesis with time of murine mammary epithelial cells in 45°C temperature shift cultures. Our cleavage oligosaccharides were also active in this heat shock protection system. Thus these oligosaccharides appear to have a generalized function in accelerating the recovery of epithelial cells from physical injury — they are cytoprotective oligosaccharides.

In conclusion these studies revealed that aloe oligosaccharides, cleaved from native polysaccharide by cellulase,' can protect CHS responses from UV-induced suppression. Aspects of these basic findings have been confirmed by other laboratories (Lee etal, 1999). This cytoprotection by cleavage oligosaccharides led us to explore if other plant complex carbohydrates, associated with a novel mechanism of response to cellular injury, could protect the cutaneous immune responses (Strickland etal., 1999).

Oligosaccharins and other therapeutic complex carbohydrates

At present, our best chemically-defined SIS therapeutic saccharide is the Tamarind-seed xyloglucan polymer made from the nonasaccharide repeating unit (Structure 3) (Strickland etal, 1999).

Structure C

Tamarind Xyloglucan Nine sugar 'monomeric' repeating unit

| | Glucose A Xylose Q Galactose

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