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activity [2]. The FT-IR profiles of different samples of A. vera studied typically indicate the presence of groups –OH (3,420 cm⁻¹), stretching of –CH (2,923 cm⁻¹), C=O stretching of acetyl (1,760–1740 cm⁻¹), C=O (1,650–1,578 cm⁻¹), COO– asymmetric stretching (1,598 cm⁻¹), CH₃ and COO– symmetric stretch (1,428 cm⁻¹), C–O–C stretching of acetyl groups (1,248 cm⁻¹), C–O–C ether in sugar (1,091–1,030 cm⁻¹) and glucan bands (1,031 cm⁻¹) [22, 28]. High-intensity peaks at 3,420 cm⁻¹ also confirm the presence of hydroxyl groups, indicating the presence of a mixture, as reported by some authors. A great band in the 1,078–1,036 cm⁻¹ range also shows the presence of polysaccharide sugars such as mannose, galactose, and units of glucans [22, 79].

Using the polysaccharide fraction Kiran & Rao [22, 37] showed the presence of peaks at 3,420, 1,740, 1,598 and 1,248 cm⁻¹ indicating the presence of –OH, C=O, COO– and C–O–C stretching of acetyl groups, suggesting that the powder of the polysaccharide fraction consists of high levels of storage polysaccharides, such as acemannan. These results have been confirmed in other recent observations of bioactive acetylated polysaccharide [5, 7, 24, 80]. The profile reported by Ray & Aswatha [24] in their study of freeze dried mucilaginous gel indicated the presence of absorption bands at 1,635 cm⁻¹ and 1,078.53 cm⁻¹. It was more marked in the gel obtained from 3-year-old plants, indicating a high level of acemannan in the sample relative to gel from 2 to 4-year-old plants.

      The profile of the IR spectra of fractions obtained from A. vera skin and gel fraction was similar, indicating similar macromolecular structures. The signals for the hydroxyl group and ether (C–O–C) in sugar units were strongly absorbed at 3,420 cm⁻¹ and 1,050 cm⁻¹, respectively. A band at 1,066 cm⁻¹ in polysaccharides from the skin and gel represents the presence of mannopyranose components. More specifically, peaks at regions in the 1,736–1,740 cm⁻¹ and 1,246–1,252 cm⁻¹ range confirm the presence of O-acetyl ester [34]. Sriariyakul et al. [30] examined dehydrated samples using FT-IR spectroscopy and confirmed the presence of bands of C=O of acetyl groups at the 1,740 cm⁻¹ wavelength. The authors reported a marked decrease in these bands and that this decrease might be attributed to the deacetylation of acemannan during the hot-air drying process combined with FIR radiation and HVEF.

      The samples submitted to the pasteurization processes in the studies of Rodríguez-González et al. [7] underwent partial deacetylation of the acemannan polymer, evidenced by a decrease in the 1,740 and 1,250 cm⁻¹ bands, which correspond to C=O and C–O–C stretching of acetyl groups. Chokboribal et al. [14] reported that complete acemannan deacetylation led to the disappearance of the absorption of the carbonyl at 1,740 cm⁻¹.

       1.4.2.3 Nuclear Magnetic Resonance Spectroscopy

      Diehl & Teichmuller [36] showed that ¹H-NMR is an essential tool for assessing the identity and quality of A. vera gel preparations. Acemannan has β1→4 linkages in the partially acetylated mannose residues at positions 2, 3 or 6, and features a characteristic signal (2.00–2.26 ppm) on ¹H-NMR spectra of acetyl groups which can be regarded as the fingerprint of A. vera. Thus, the structure and position of the functional groups of acemannan can be analyzed by ¹H-NMR spectroscopy, constituting an essential means of identifying and preventing falsification by checking for the presence of acetyl groups [14, 19, 36]. Chokboribal et al. [14] stated the data derived from ¹H-NMR and IR spectra, together with the ¹³C-NMR spectra, confirmed that the precipitate isolated from A. vera gel as acemannan. The ¹H-NMR results also indicate that approximately 95% of mannose residues were acetylated, that protons at C2–C6 (H_2-6) occurred at 3.1–4.0 ppm, whereas H_Ac protons occurred at around 1.9 ppm.

      Davis & Goux [38] studied commercially available products and, comparing with alcohol-precipitated polysaccharide fractions of A. vera, showed high resonance of methyl acetyl protons at 2.0 and 2.2 ppm. Spectra also revealed signals of protons from the pyranose ring H₂–H₆ at 3.0–4.2 ppm and acetic acid methyl protons at 1.9 ppm.

According to Ray & Aswatha [24], the signals corresponding to acemannan and glucose are evident in the ¹H-NMR spectra. Peaks at 2.05 and 5.13 ppm suggest the characteristic presence of acemannan and glucose, respectively. In the present analysis, the malic acid peak at 3.08 ppm, and a high concentration of phenolics may have suppressed the malic acid peak at 4.3 ppm.

      In an analysis of 21 commercial A. vera gel products, Kim et al. [32] found that 33% of the samples contained high levels (45–95%, w/w) of maltodextrin as an adulterant undeclared on labels. In another study, a sample exhibited a signal at 5.4 ppm on ¹H-NMR spectra, revealing significant amounts of maltodextrin, although in this case, the presence of the adulterant was declared [19].

      A. vera gel contains three principal components: acetylated polysaccharides (acemannan), glucose, and malic acid. High levels of lactic acid and acetic acid in products 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

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