(–) samples are not subjected to hydrolysis
Chokboribal et al. [14] also used the acid hydrolysis method to determine the composition of monosaccharides, structure and molecular mass of the alcohol precipitated polysaccharide performing these analysis using liquid chromatography with reflective index detector, Nuclear Magnetic Resonance Spectroscopy (1H NMR), Fourier Transform Infrared Spectroscopy (FT-IR), and size-exclusion chromatography, respectively. The data obtained confirmed the polysaccharide was acemannan.
The presence of uronic acids together with trace levels of arabinose and ramnose is indicative of residual amounts of pectinic polysaccharides in the polysaccharide fraction, as previously observed in the hydrosoluble extracts from A. vera leaf pulp [4, 9].
1.4.2 Analytical Techniques
HPLC is a technique used for fractionating, purification and determining the size of molecules. When separation is based on molecule size, these techniques are referred to as High Performance Size Exclusion Chromatography (HPSEC), using specific chromatographic columns and known size standards (dextran or pullulan). The detectors employed were refraction index (RI) [14, 21, 75, 78] and Multi-angle Laser Light Scattering (MALLS) [21, 75, 78]. HPAE-PAD was also reported, consisting of a separation technique based on the charge of molecules and useful for separating neutral molecules.
Gas Chromatography is another widely employed technique suitable for analyzing volatile compounds. Other commonly used analytical techniques include Infrared Spectroscopy (IR), Nuclear Magnetic Resonance (NMR), and Mass Spectrometry (MS). These methods can be used to investigate the structure of a compound or to identify a compound based on the specific signatures of a chemical bond or group present.
UV/Vis studies were also found as a complementary technique for characterizing compounds, in which a marker such as chromophore was used. Recently, using a microarray-based technique, it was possible to study the relative abundance, and interactions between hundreds or thousands of molecules simultaneously, using very small volumes of A. vera extracts of different species (Table 1.2).
1.4.2.1 Chromatography Analysis
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 ImmunostimulantTM 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%.
1.4.2.2 Infrared Spectroscopy (IR)
Fourier-transform infrared spectroscopy (FT-IR) spectra have been widely used for comparisons in the distribution of functional groups of A. vera products. Absorption bands at 1,247 cm−1