Читать книгу Polysaccharides - Группа авторов - Страница 21

Femenia et al. [9] used a HPLC method with a RI detector to quantify the free sugars in different parts of A. vera leaf and found that glucose accounted for 95% of the total monosaccharides analyzed. For the composition of carbohydrates of the polysaccharides hydrolyzed with sulphuric acid, the predominant sugars were mannose and glucose in all fractions, representing 55–75% of the total monosaccharides determined. Mannose residues in the fillet and gel were probably from acemannan, the main component of A. vera, where large amounts of acemannan were found in these tissues [8]. After elution on a specific size-exclusion column using HPLC-RI, Medina-Torres et al. [72] reported that the molecular weight of dry mucilage in a spray dryer was determined as 4.18 × 10⁴ Da and for fresh mucilage as 5.96 × 10⁴ Da. The reduction in molecular weight is expected owing to the heat treatment and high shear forces present in the spray drier chamber.

Chokboribal et al. [14] reported that the mean molecular weight of the acemannan isolated, calculated based on its retention time, after analysis by HPLC using a size-exclusion column with RI detectors was 190–220 kDa. Campestrini et al. [13], also using size-exclusion chromatography calibrated against dextran standards with a MALLS detector, observed a large peak at approximately 38 min for both the samples from the crude extract of A. vera and for the polysaccharide fraction, corresponding to a high molecular weight compound, identified as a partially acetylated glucomannan whose molecular weight was determined at 1.2 MDa (1,200 kDa).

A marked increase in molecular weight of purified acemannan was observed with an increase in temperature using hot-air drying in a study performed by Femenia et al. [20]. The sample dehydrated by freeze drying had an estimated mean molecular weight of 45 kDa, whereas the samples dries at 70 and 80 °C had weights of 75 and 81 kDa, respectively. The difference is explained by loss of galactosyl residues, which together with the deacetylations observed may have contributed to interactions between different mannose chains by hydrogen bridges, resulting in chains rich in mannose of high molecular weight.

Turner et al. [21] analyzed 32 commercially available products, fresh A. vera and Acemannan Immunostimulant^TM by size-exclusion chromatography (SEC) calibrated against pullulan standards with RI detector and MALLS. They reported that when using SEC/RI, both the column and mobile phase selections affected the determination of molecular weights, and also advocated the need to use a complex carbohydrate similar to acetylated mannan from A. vera as a comparative standard, given these are the most abundant polysaccharides in the plant. SEC/RI will not yield reliable data for molecular weight calculations unless there are peaks with good resolutions where the apex cannot be seen. Consequently, molecular weights using SEC/RI are not acceptable by regulatory agencies. But molecular weights using MALLS are required by the FDA (Food and drug administration). Of the two methods, MALLS promote more defensible data on molecular weights and particle size.

He et al. [78] have also conducted studies using HPSEC with RI detector and MALLS, comparing to calibration curves with dextran and pullulan standards. The results have shown that there is a considerable difference between the distinct methods due to different structures, the composition of the standards, and interactions between polysaccharides. It is also reported that it is hard to determine the molecular weight of unknown polysaccharides using only one standard due to the complexity of polysaccharides found in Aloe vera, suggesting MALLS as the method of choice.

Bozzi et al. [19] showed that Acemannan Hydrogel^™ is composed of a high molecular weight component with a mean molecular weight of 3.7 MDa, and another low molecular weight component of 27.4 KDa using ion-exchange chromatography. Immuno-10 showed a range of approximately 10.0 KDa to 1.0 MDa with a prominent peak at 39.0 kDa. Minjares-Fuentes et al. [27] used Gas Chromatography with a flame ionization detector (GC-FID) to detect the composition of sugars in their samples after acid hydrolysis at 100 °C. Mannose was the predominant sugar, at around 70%, whereas galactose corresponded to 29.4% and glucose, 0.6%.