Polysaccharides. Группа авторов

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[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 indicate bands of C–O–C of acetyl groups, cited by many authors as the component that promotes biological 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].

      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 (H2-6) occurred at 3.1–4.0 ppm, whereas HAc 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.

      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

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