Detection of Seasonal Variation in Aloe Polysaccharides Using Carbohydrate Detecting Microarrays

Abstract

Aloe vera gel is a globally popular natural product used for the treatment of skin conditions. Its useful properties are attributed to the presence of bioactive polysaccharides. Nearly 25% of the 600 species in the genus Aloe are used locally in traditional medicine, indicating that the bioactive components in Aloe vera may be common across the genus Aloe. The complexity of the polysaccharides has hindered development of relevant assays for authentication of Aloe products. Carbohydrate detecting microarrays have recently been suggested as a method for profiling Aloe polysaccharide composition. The aim of this study was to use carbohydrate detecting microarrays to investigate the seasonal variation in the polysaccharide composition of two medicinal and two non-medicinal Aloe species over the course of a year. Microscopy was used to explore where in the cells the bioactive polysaccharides are present and predict their functional role in the cell wall structure. The carbohydrate detecting microarrays analyses showed distinctive differences in the polysaccharide composition between the different species and carbohydrate detecting microarrays therefore has potential as a complementary screening method directly targeting the presence and composition of relevant polysaccharides. The results also show changes in the polysaccharide composition over the year within the investigated species, which may be of importance for commercial growing in optimizing harvest times to obtain higher yield of relevant polysaccharides.

Keywords: Aloe; authentication; carbohydrate detecting microarrays; plant cell walls; polysaccharides; seasonal variation; succulent tissue.

FIGURE 1

Schematic presentation of the CoMPP method illustrated with Aloe arborescens. Species are sampled in triplicates and prepared for extractions. The supernatant from each sequential extraction step is printed separately in technical replicates on nitrocellulose membranes. The printed microarrays are then probed with a selection of molecular probes.

FIGURE 2

All extractions are summarized per season. The Ph. Eur. Material extractions (standards) are also summarized for each species. All tested antibodies are included. Included antibodies and their target epitope as well as the origin of the antibody and where their bindings have been described is listed below the heatmap. DE, degree of esterification; RGl, rhamno-galacturonan; KG, kyloglycan; Neg. R, anti-rat; Neg. M, anti-mouse. The highest mean value of the entire dataset was assigned the value of 100%, and the remainder of the data were adjusted accordingly and normalized with a 5% cut off (represented with a zero – “0”).

FIGURE 3

Anatomy of four Aloe species, (A) Leaves and (B) cross-sections from left to right of Aloe vera, A. vaombe, A. arborescens and A. decaryi. (C) Cross-sections from left to right of Aloe vera, A. voombe, A. arborescens and A. decaryi stained with Toluidine blue. Marked are ep, epidermis; olm, outer leaf mesophyll; v, vasculature; ilm, inner leaf mesophyll.

FIGURE 4

Immunolabeling using the mannan specific monoclonal antibody BS-400-4 (red channel). Overlay with CalcofluorWhite (blue channel) counterstain to visualize cell walls.

FIGURE 5

Immunolabeling using the mannan specific monoclonal antibody LM21 (red channel). Overlay with Calcofluor White (blue channel) counterstain to visualize cell walls.

FIGURE 6

Species specific heatmaps and graphs showing the changes in mannan over time in the three sequential extractions. RGI, rhamno-galacturonan; XG, xyloglycan; Meg. R, anti-rat; Neg. M, anti-mouse. The highest mean value of the entire dataset was assigned the value of 100%, and the remainder of the data were adjusted accordingly and normalized with a 5% cut off (represented with a zero – “0”).