Annotation of the results obtained in 2022
At the stage of the reporting period 2022-2023, the process of isolating hemicelluloses from hardwood (aspen) and coniferous (spruce, larch) wood was optimized. Hemicelluloses have been produced for further chemical modification. The oxidative non-catalytic delignification of aspen wood in the medium "acetic acid - water - hydrogen peroxide" was studied. The maximum yield of hemicellulose (up to 9.678 wt.%) is achieved at a process temperature of 100 °C and a duration of 4 hours. With the help of mathematical processing, the optimal conditions for the process of delignification of aspen wood were determined - the temperature of the process is 98.8 ° C, the duration is 3.51 hours. The obtained hemicelluloses were characterized by a complex of physicochemical methods: GPC, FTIR, NMR, GC, TGA. The composition and structure of hemicelluloses were determined, as well as the effect of isolation conditions on product characteristics. It has been established that aspen wood hemicelluloses isolated under the conditions chosen by us have different molecular weight distribution and weight average molar mass (8.932 – 33.142 g/mol). With an increase in the process temperature from 70 to 100 °C, PD decreases from 6.18 to 2.05, indicating an increase in the homogeneity of the HC. The isolated hemicelluloses with a high degree of depolymerization consist mainly of glucuronoxylan and contain a small amount of residual lignin. It was confirmed by HSQC that β-d-Xylp is the basis of aspen wood hemicelluloses, which contain a linear xylan main chain linked by β-(1→4) glycosidic bonds with small amounts of hexose, mannose and uronic acid units in the side chains. Solvents were screened for the reaction of spruce galactoglucomannan sulfation with sulfamic acid in the presence of the main catalyst, urea. It was found that the degree of sulfation increases in the series morpholine < piperidine < pyridine < diglyme < dimethylformamide (DMF) < 1,4-dioxane < dimethyl sulfoxide (DMSO). However, due to the complexity of the process of purification of the product from DMSO, the optimal solvent is 1,4-dioxane The influence of the duration of the sulfation process on the molecular weight characteristics of the products and the degree of sulfation has been studied. A correlation was found between the weight average molecular weight and the degree of sulfation, which generally indicates the non-destructive nature of the selected sulfation process. However, after reaching a maximum after 120 minutes of the process, both in terms of molecular weight and sulfur content (21524 g/mol and 12.67 wt.%, respectively), an increase in the reaction time causes hydrolysis of glycosidic bonds, which leads both to a decrease in the molecular weight of the product and a decrease in degree of sulfation. The structure of sulfated galactoglucomannan was studied by IR spectroscopy. The introduction of sulfo groups into the galactoglucomannan molecule is confirmed by the appearance of absorption bands at 1255 and 875 cm-1 corresponding to antisymmetric stretching vibrations of SO2 υas(O=S=O) and stretching vibrations of С–О–S υ(С–О–S) groups. The band with a maximum at 1029 cm-1 refers to symmetric stretching υs(O=S=O) vibrations. The bending δ(O=S=O) and stretching υ(S–O) vibrations correspond to two low-frequency bands with maxima at 671 and 528 cm-1. A new environmentally safe method for modifying larch hemicellulose, arabinogalactan (AG), with polybasic carboxylic acids (citric, succinic, oxalic, and adipic) has been developed to obtain composite materials. The synthesized derivatives of AG were studied by a complex of physicochemical methods, including gel permeation chromatography (GPC), infrared Fourier spectroscopy (FTIR), thermogravimetric analysis (TGA), X-ray diffractometry (XRD), scanning electron microscopy (SEM)), a study of sorption capacity was carried out. It has been shown that heat treatment leads to the formation of additional inter- and intramolecular bonds between carboxylic acids and polysaccharide molecules. The formation of ester bonds is confirmed by the appearance of absorption bands in the IR spectra in the region of 1750–1690 cm-1. With the help of a TGA study, it was found that the most thermally stable (up to 190 °C) sample is arabinogalactan oxalate obtained by heat treatment. The SEM study of the synthesized AG films showed that the modified samples have a uniform film surface provided by crosslinking. When studying the sorption properties of AG derivatives, it was found that AG succinate (82.52 %), obtained by lyophilization, has the highest sorption capacity due to the developed mesoporous surface, which, in turn, makes the synthesized films promising environmentally friendly materials for use as drug carriers, sorbents and water treatment products. The process of formation of hemicellulose/chitosan and sulfated hemicellulose/chitosan polyelectrolyte complexes by the drop method with variation of the polyanion/polycation ratio has been studied. Under the condition of adding a solution of chitosan to sulfated hemicellulose, PECs with a negative surface charge were obtained, as evidenced by the ζ-potential from -11.1 to -30.1 mV. However, as the content of chitosan increases, the ζ-potential acquires positive values (from 14.7 mV), which do not ensure the stability of PEC. When changing the mixing order (chitosan/CHC), particle aggregation was observed, which indicates the influence of the mixing order during the formation of PEC. The antioxidant and anticoagulant activity of hemicelluloses and their derivatives was studied. The study showed that aspen hemicelluloses have a strong inhibitory effect on DPPH radicals and a dose-dependent pattern is observed. The activity of all polysaccharides in neutralizing DPPH and hydroxyl radicals was lower than the absorption capacity of vitamin C (Vc) at all tested concentrations (0.5; 2; 5 mg/mL). The effect of antioxidants on DPPH scavenging activity was found to be their hydrogen donating capacity, i.e. low molecular weight polysaccharides with high uronic acid content had a stronger hydrogen donating capacity resulting in better DPPH radical scavenging activity resulting in greater solubility. and easier interdiffusion to transfer a greater ability to donate hydrogen. Samples containing more phenolic components (70-4, 80-4) show a stronger ability to remove hydroxyl radicals, however, in terms of the purity of the main product, they are inferior to samples (100-4 - 100-1). Samples of sulfated galactoglucomannans with various degrees of sulfation were evaluated in experiments on human platelet aggregation and human erythrocyte hemolysis. The investigated derivatives of galactoglucomannans in vitro at concentrations up to 0.02 mg/ml did not affect: the time of appearance of a human blood clot in the blood recalcification time test; at the time of the appearance of a human plasma clot in the activated partial thromboplastin time test; on hemolysis of human erythrocytes (the degree of hemolysis is less than 2 %). It has been shown that sulfated derivatives of galactoglucomannans, which do not affect the coagulation of human blood/plasma and the erythrocyte membrane, can be used as potential components of the developed constructs for the delivery of drugs or genes.

 

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Annotation of the results obtained in 2023
At the stage of the reporting period 2023-2024, further chemical modification of hemicelluloses from coniferous wood was carried out. Anionic derivatives of hemicelluloses have been produced for further chemical modification. The process of oxidation of AG with the TEMPO/NaBr/NaOCl system was studied, resulting in the production of polyuronides with improved functional properties. It has been shown that the maximum uronide component lasts for 3 hours and does not change thereafter. Using gel permeation chromatography (GPC), the non-destructive nature of the oxidation process was established, as well as a proportional increase in molecular weights during the process, which is reflected in an increase in molecular weight (up to 15,700 g/mol). Using IR-Fourier spectroscopy, characteristic absorption bands (1742, 1639 and 1403 cm-1) were established, indicating the occurrence of oxidative processes with subsequent reduction of the carboxyl group. Based on the TGA/DTG curves, it was shown that oxidized AG-TH has higher thermal stability compared to native AG due to the modification process. An assessment of the flocculation ability showed that even a low concentration of interacting AG-TH samples increases the flocculation rate of bentonite clay in the presence of iron and calcium cations. The indicators of antioxidant activity of AG are generally characteristic of polyuronic acids, which indicates the preservation of the inherited properties of native AG. The oxidation process of galactoglucomannan in the TEMPO/NaBr/NaOCl system was studied. Based on data on the consumption of NaOH during the process, it was revealed that the process of GGM oxidation occurs in stages, with variable dynamics, which is consistent with the assessment of the uronide component of the hydrolysates of the reaction medium of HGM oxidized by the “carbazole” method. During the oxidation of GGM, its uronide component increases from 14.8 to 38.9%, which is achieved as a result of a 5-hour reaction time. Using IR spectroscopy, a redistribution of absorption bands in the region of 1730 cm-1, corresponding to vibrations of alkyl esterified carboxyl groups, was established. Decrease in intensity of p.p. in the oxidized product indicates that HGM undergoes a series of rearrangements of some alkyl substituents to others due to better orientation in a polar environment during the oxidation process. A new environmentally friendly method has been developed for modifying spruce hemicellulose—galactoglucomannan (GGM)—with polybasic carboxylic acids (citric and succinic) under various conditions (without/using a plasticizer and catalyst) to obtain new functional materials. It has been shown that heat treatment at elevated temperatures (150 °C) leads to the formation of additional inter- and intramolecular bonds between the carboxylic acid groups of carboxylic acids and the hydroxyl groups of polysaccharides. Using GPC, it was found that the esterification reaction resulted in modified samples with significant changes in hydrophobicity due to the ester bonds formed. Galactoglucomannan citrates exhibited greater hydrophobicity compared to succinates. The formation of ester bonds is confirmed by the appearance in the IR spectra of absorption bands in the region of 1720–1600 cm–1, corresponding to the stretching vibrations of the C=O group, as well as a signal in the region of 1200 cm–1, characteristic of vibrations of the C-O group of carboxylate. Electron microscopy data have established that the modified samples have a uniform and smooth film surface, which is explained by the smooth removal of the solvent from the system followed by high-temperature drying, making it possible to obtain a fairly dense and uniform material. Based on the results obtained, the synthesized films are promising environmentally friendly materials for use as carriers and shells of medicines, as well as packaging materials. The multistage mechanism of sulfation of galactoglucomannan by the sulfamic acid-urea complex in dioxane medium was confirmed. It has been established that the sulfation of spruce galactoglucomannan and fir galactoglucomannan occurs by similar mechanisms. The obtained samples of sulfated spruce galactoglucomannan have a narrow MWD, which confirms the homogeneity of the samples. The sulfur content in sulfated galactoglucomannans increases in proportion to the increase in process duration in the range of 30-150 min, while the degree of polydispersity decreases due to a decrease in the proportion of low molecular weight fractions of GGM, which are more susceptible to sulfation. This dependence allows us to obtain sulfated derivatives of GGM with a given sulfur content. Based on the results of the research, pilot batches of sulfated GGM spruce were produced for further study of their biological activity, as well as for use as components of interpolyelectrolyte complexes. The process of formation of polyelectrolyte complexes (PEC) “sulfated hemicellulose/chitosan” by the drop method with varying the polyanion/polycation ratio was studied. For the synthesis of PEC, sulfated derivatives of GGM with a high degree of sulfation SGGM-150 Mw 12500 g/mol, PD 1.62 and sulfur content 12.9% were used. By adding a chitosan solution to sulfated hemicellulose, PECs with a negative surface charge were obtained, as evidenced by a ζ-potential from -12.6 to -34.4 mV. However, with an increase in the chitosan content, the ζ-potential acquires weak positive values (up to 20.9 mV), which do not ensure the stability of the nanoparticles. When the mixing order was changed (chitosan/SGGM), particle aggregation was observed, which indicates the influence of the mixing order on the formation of NPs. The antioxidant properties of oxidized hemicelluloses have been studied. The IC50 values for the AG-T sample, based on both DPPH and hydroxyl radical scavenging (68.5 and 2.1 mg/mL, respectively), were found to be different for AG-TH (15.1 and 2.7 mg /ml, respectively) is insignificant. However, the protonated form of AG-TH has a higher ability to inhibit DPPH radicals, which is associated with a significantly higher mobility of H+ ions compared to Na+ ions in the AG-T sample. However, the differences in inhibition of hydroxyl radicals are small and within the limits of experimental error.

 

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