are the result of bacterial degradation, hydrolyzes, or thermal degradation of the materials during production or storage [39].
Malic acid is the only organic acid present in fresh A. vera gel, constituting a natural component essential for the plant’s photosynthesis. By contrast, commercial products may contain high levels of other organic acids, such as citric acid, lactic acid, and succinic acids [19].
In the studies by Minjares-Fuentes et al. [27], the 1H-NMR spectra exhibited signals corresponding to malic acid and acemannan polymer, two natural components found in A. vera gel, in all samples studied. Additionally, the quantitative analyses performed to determine the levels of acetylation of acemannan showed a 40–70% reduction in acetylation of the polymer depending on the drying process used. The deacetylation of acemannan was also observed on FT-IR analyses by other authors [28, 30].
Citric acid is a natural preservative added to foods as a flavor enhancer and to prevent oxidation that is widely used in the food industry. The pH of juice from A. vera gel is generally adjusted to 3.0–3.5 with citric acid before concentration and drying. However, some samples contained extremely high levels of citric acid [19]. Acemannan can be converted into acetic acid (2.08, 11.00 ppm), lactic acid (1.34; 2.00; 4.27; 11.00 ppm), and succinic acid (2.5; 11.00 ppm) by microbial contamination and subsequent degradation. Detection of these organic acids on 1H-NMR spectra suggests the occurrence of degradation [24] and should be absent in quality A. vera products.
The results found by Minjares-Fuentes et al. [27, 28] showed that acemannan polymer was severely affected by the different drying methods employed, with acemannan deacetylation detected by 1H-NMR and FT-IR techniques, having a potentially serious impact on the biological activities attributed to the plant. Acetylated polysaccharides have been identified as an authentic marker of A. vera gel, and good quality derivatives should contain high levels of these polysaccharides [39].
1.4.2.4 Mass Spectrometry
The MS technique [39], in conjunction with NMR, can yield data elucidating molecular structures. Simões et al. [40] conducted the first study on the structural characteristics of acemannan using the technique to provide an acetylation profile of commercially available bioactive acemannan and was first to observe the presence of arabinose residues in this structure. Electrospray ionization mass spectrometry (ESI-MS) and Tandem Mass Spectrometry (MS/MS) of oligosaccharides from acemannan showed that the molecule was highly acetylated. This mannan contained, on average, two acetyl groups per sugar unit, double that reported by other authors.
1.4.2.5 Ultraviolet–Visible Spectroscopy
UV-Vis spectroscopy technique is applied for quantitative analyses of glucose and mannose using a wavelength of 490 nm after acid hydrolysis of acemannan with appropriate standards for each monomer [22, 37, 60, 61, 69], as well as for quantitative analyses of acemannan at the wavelength of 540 nm [24, 37, 68, 73]. Eberendu et al. [68, 81] developed a quantitative colorimetric method for measuring glucomannan from A. vera without previous separation or chemical degradation of the polymer. These colorimetric assays are based on the colored complex formed by binding the β (1–4)-linked polysaccharides and dye Congo red (sodium 4,4’-diphenyl-2,2’-diazo-bis-1-naphtlamino-4-sulfonate).
The maximum wavelength of light absorption of Congo red in aqueous solution at 1% (w/v) is approximately 488 nm and maximum absorption of Congo red when conjugated with acemannan shifts the wavelength to 540 nm. The linearity obtained by the plot of absorption readings versus concentration (mg/L) of polysaccharides had a correlation coefficient of 0.999 at concentrations of between 0.9 and 72.7 mg/L. This result confirmed that the colorimetric assay method developed has many advantages over any currently used method for measuring A. vera polysaccharides, and that the assay is accurate for measuring the true amount of glucomannan and is not subject to interference from other components [68].
To date, only a few researchers have documented the quantitative analysis of acemannan by chromogenic formation of Congo red–acemannan [82]. Acemannan was described as the only polysaccharide able to form a characteristic conjugate by reacting with Congo red stain in a basic medium, and the increase in absorption to a wavelength of 540 nm is stoichiometric in relation to acemannan concentration [83].
Ray & Aswatha [24], also using the spectrophotometric technique, detected that the absorbance of the Congo red–acemannan conjugate varied with age of the plant and season of the year. The authors showed that 3-year-old plants harvested in the summer produced mucilaginous gel containing a greater amount of acemannan, given it had higher absorbance of the chromophore compared to 2-year-old plants and was higher than for plants harvested in winter and the rainy season.
The International Aloe Science Council (IASC) has been approved the Congo red assay for quantifying the content of glucomannans from A. vera and its derivates. However, the A. vera gel content other polysaccharides with β(1–4)-linked and could generate confusion or false positive [33]. Metcalfe [84] developed and validated a simple and inexpensive method, where the acetyl groups of the acemannan polysaccharide are converted into a quantified ferric-acetohydroxamic complex using a UV–Vis spectrophotometer at 540 nm.
1.4.2.6 Comprehensive Microarray Polymer Profiling
Ahl et al. [85] presented a new methodology using a microarray-based technique (CoMPP) for the determination of Aloe polysaccharides in different Aloe species. For them, the CoMPP technique is a useful tool for the characterization of Aloe polysaccharides.
1.5 Conclusion
A large variety of different methods of obtaining A. vera gel involving more than one step was found. Products derived from A. vera gel are commercially available in the form of gels, liquids, and particularly in spray dried or freeze dried products extracted from the inner part of the leaf, followed by stages of separation of insoluble fibers, alcohol precipitation of polysaccharides and purification of acemannan. Several authors have analyzed the composition of free sugars and used techniques for separation and purification of polysaccharides from A. vera gel and have determined the constitution of the monomers present. However, the types and molecular sizes of the polysaccharides isolated from A. vera gel vary due to factors such as the plant subspecies or seasonal differences in cultivation, different techniques used to isolate the polysaccharide, polysaccharide degradation during processing and also the analytical methodology applied.
These differences hamper standardization of the process of obtaining A. vera gel and of an analytic methodology for quantifying its principal polysaccharide, acemannan, yielding non-reproducible data. However, based on the studies reviewed, natural polysaccharides are not stable under conditions of stress, such as the application of heat or enzymatic processes, leading to degradation of acemannan and loss of physical and biological properties of A. vera. Thus, the method by which the plant is processed can lead to final preparations with different chemical compositions. Therefore, selecting the right process to ensure the desired chemical composition of the end product is vital, thereby retaining the high molecular weight polysaccharides present in the fresh gel.
Given that the inner gel obtained from A. vera is considered a source of biological activity attributed to the plant, it is essential to apply the body of knowledge on the chemical composition of A. vera to inform future studies for product standardization and development, and also to establish the most suitable method (or combination of methods) for quality control and subsequent confirmation of pharmacologic activity.
References
