Xylan from Dinizia excelsa: Chemical characterization and biological activities

Abstract

Dinizia Excelsa is an Amazonian tree with a wide range of applications as a raw material in the industry. The objective of this study was to extract, characterize, and evaluate the biological activities of xylan extracted from Dinizia excelsa wood. The xylan was obtained in five stages, including delignification, precipitation, purification, and freeze-drying. The physicochemical analysis of xylan included the determination of monosaccharides, elemental composition, FTIR analysis, 2D nuclear magnetic resonance spectroscopy, and the determination of molecular weight. Xylan had an extraction yield of 28.44% and an elemental composition of 35.03% carbon, 5.65% hydrogen, and 59.32% oxygen. FTIR analysis revealed similarities between Dinizia excelsa xylan and commercial xylan. 2D NMR analysis confirmed the presence of characteristic xylan groups. Furthermore, xylan has a low molecular weight. In vitro cytotoxicity tests demonstrated low toxicity, indicating its potential for biological applications. Immunomodulatory activity assays revealed that xylan stimulated cell proliferation and the production of anti-inflammatory cytokines. The anticoagulant activity of xylan was low compared to heparin. The antioxidant activity of xylan was weaker compared to ascorbic acid and butylated hydroxytoluene (BHT). These results indicate that xylan from Dinizia excelsa has potential for several biomedical applications due to its immunomodulatory and anticoagulant properties.

Key words Dinizia excelsa; Amazonian tree; Xylan; immunomodulatory

INTRODUCTION

Dinizia excelsa, an Amazonian tree popularly known as Angelim Vermelho, Angelin Pedra, and Faveira de Ferro, is a member of the Fabaceae family and can reach a height of 55 m (Melo & Varela 2006, Mesquita et al. 2007). The wood possesses high mechanical strength and durability, in addition to being resistant to attacks from xylophagous agents. These characteristics have allowed this wood to be used as a product in civil construction, as well as in the construction of decks, beams, floors, and other engineering works (Melo & Varela 2006, Conceição et al. 2021). Although its application in the wood sector is known, its pharmacological potential has not been fully elucidated. This is because its extractives and macromolecules have not been chemically characterized, which is a crucial step to determine its biotechnological applications.

Among the macromolecules are xylans, which are polysaccharides with great potential for study and biomedical use. Xylans are a group of polysaccharides with a common structural chain in the β-(1→4) conformation, which is associated with a xylose residue (Duan et al. 2021, Curry et al. 2023, Cyran et al. 2024). Hemicelluloses are a group of complex carbohydrates, being considered one of the polysaccharides most abundant in the plant cell wall, second only to cellulose. Xylose polysaccharides provide mechanical strength, regulate the osmotic pressure of cells, and protect the wood against pathogens (Curry et al. 2023).

The literature presents three applications of xylans. These can be raw materials for biofuels, used as biomaterials, or have biomedical applications (Curry et al. 2023, Teli et al. 2024). As a biofuel, xylans can add value to cellulose industry waste, which is rich in xyloses. As a biomaterial, this product can be utilized for producing bioplastics, hydrogels, biofilms, and as a carrier for drugs and other substances due to its biodegradability (Nechita et al. 2021). Xylans also exhibit antimicrobial, prebiotic, antitumor, and immunomodulatory activities (Costa Urtiga et al. 2020, Oliveira et al. 2024, Premarathna et al. 2024).

Due to the versatility of applications, the global market for xylan-derived products was valued at 1.66 US billion in 2022 and is expected to grow by 6% between 2023 and 2029, reaching approximately USpf-word suggestion 2.50 billion (Xylan Market 2024). These numbers indicate that xylans are a renewable raw material of great importance for the sustainable and high-value-added products sector.

In this context, tthis work aims to conduct, for the first time, a preliminary characterization of xylan obtained from Dinizia excelsa wood. In addition to evaluating cytotoxic, hemolytic, anticoagulant, antioxidant, and immunomodulatory activities in vitro. This work aims to contribute to the valorization of bioactive macromolecules obtained from the Amazon forest.

MATERIALS AND METHODS

Plant

Dinizia excelsa wood was provided by the company Mil Madeiras Preciosas Ltda (PRECIOUS WOODS), located in Itacoatiara, Amazonas, Brazil, known for its sustainable practices in forestry management. The species was registered in SisGen with the number ACEE312. After drying at 60 °C for 48 h, grinding and sieving (0.11 mm), the material was stored at 30 °C.

Process of obtaining xylan

The xylan was obtained in five different stages following the methodology proposed by Cruz Filho et al. (2023). Initially, the milled wood was dried at 80 °C for 24 h to remove moisture. Delignification was conducted using a ratio of 1:10 (w/v) in 500 mL of water, 35 g of sodium chlorite, and 2.5 mL of glacial acetic acid at 60 °C for 3 h. The reaction was carried out in a 1 L flask. At the end of the process, the system was filtered, and the solid obtained was dried at 80 °C for 24 h (Step I).

To the delignified material (34.6 g), a solution of 10% (w/v) sodium hydroxide (NaOH) and 1% (w/v) boric acid (H₃BO₃) (346 mL) was added. The mixture was stirred at 200 rpm at 60 °C for 3 h (Step II). The system was filtered, and xylan was precipitated with ethanol (95%, v/v) containing acetic acid (10%, v/v), then incubated at 4 °C for 12 h (Step III). Impurities were removed by washing with ethanol (95%, v/v), and the suspension was centrifuged at 10,000 g for 10 min to recover xylan (Step IV).

Finally, the xylan was resuspended in 200 mL of ultrapure water, dialyzed into 4 L of ultrapure water three times, lyophilized, and stored at 25 °C (Step V). The yield was calculated using Equation 1.

$$\text{Yield}(\mathrm{\%})=(\frac{\text{Xylan mass obtained (g)}}{\text{Total mass of dry material (g)}})\ast 100\mathrm{\%}$$ (1)

Physicochemical characterization of xylan

Analysis of the composition of monosaccharides and degradation products was carried out according to Cruz Filho et al. (2023) with modifications. Initially, 30 mg of xylan was hydrolyzed in 1 mL of 1% phosphoric acid for 8h. At the end of the reaction, the hydrolysate was filtered through 0.22 µm membranes and analyzed using a high-performance liquid chromatograph.

The identification and quantification of the compounds present in hydrolysate conducted using on a high-performance liquid chromatograph (Agilent, series 1100), using an Aminex HPX87H column (Bio-Rad) at a temperature of 60 °C. The mobile phase consisted of 5 mM sulfuric acid (H₂SO₄), with a flow rate of 0.6 mL/min. A refractive index detector was utilized for the identification and quantification of different monosaccharides. The concentration of degradation products was determined using a reverse-phase column (C-18) from Agilent Technologies. The mobile phase consisted of an acetonitrile-water-acetic acid solution (1:8:1%), and detection was done using a UV/Vis detector (274 nm) at 25 °C.

Elemental analysis of xylans (20 mg) was performed in triplicate using a CHNS/O elemental analyzer model Perkin Elmer 2400 series II. This analysis was carried out with the aim of determining the carbon (C), hydrogen (H), and nitrogen (N) contents. The oxygen (O) content was calculated by difference, subtracting the percentages of carbon and hydrogen from 100%.

The functional groups present in the xylan structure were identified using FTIR. FTIR spectra were acquired on the Shimadzu IR Prestige-Z1 spectrometer, using KBr pellets. Measurement conditions included 64 scans, a wavenumber range of 400 - 4000 cm^-1 and a resolution of 4 cm^-1.

2D NMR, HSQC (Heteronuclear Single Quantum Coherence) spectra were obtained using 30 mg of xylan dissolved in deuterated water heated to 60 °C. After dissolution and cooling, the samples were analyzed on a Bruker Avance 400 MHz spectrophotometer at 20 °C.

The molecular weight of xylan was determined using gel permeation chromatography (GPC). GPC analyses were conducted on a PL-GPC 110 system with two Plgel 10 µm 300 × 7.5 mm columns, protected by a Plgel 10 µm precolumn, at 70 °C. The eluent used was N,N-dimethylacetamide (DMA) with 0.1 M LiCl at a flow rate of 0.9 mL/min. Xylans (2.0 mg) were dissolved in 70 µL of DMA with 10% LiCl and then diluted to a concentration of 0.4%. Calibration of the GPC columns was performed with pullulan standards (800 to 10,000 Da). The detector used was a refractive index detector.

Cytotoxicity assays

The in vitro assessment of cell viability in 96-well plates was performed using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) colorimetric method. This method is based on the conversion of tetrazole salts into formazan crystals by active mitochondrial dehydrogenases, indicating viable cells. The tests were carried out according to the methodology described by Cruz Filho et al. (2023).

The cells (MJ774 macrophages, HepG2 hepatocytes, V79 fibroblasts, and Vero cells) were plated at a concentration of 2 x 10⁵ cells/mL in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% antibiotics (penicillin and streptomycin). The xylan was previously dissolved in distilled water, filtered through a 0.22 µm membrane, and diluted in RPMI medium at concentrations ranging from 500 to 7.8 µg/mL. Subsequently, it was added to a 96-well plate (100 μL/well). The plates were incubated for 72 h in a 5% CO₂ incubator at 37 °C. Then, 25 µL of the MTT solution (0.5 mg/mL) was added, and the plates were incubated for 3 h.

After 3 h of incubation, the plates were centrifuged at 4500 rpm for 10 minutes. The supernatant material was discarded, and 100 µL of DMSO was added to dissolve the formazan crystals. Finally, the microplate was read using a spectrophotometer at 570 nm. Commercial xylan was used as an experimental standard under the same conditions as the xylan obtained in this study. The positive growth control consisted of only the culture medium and cells. viability was determined by Equation 2.

$$CV(\mathrm{\%})=(\frac{\text{test absorbance}}{\text{control group absorbance}})\ast 100\mathrm{\%}$$ (2)

Cell viability data for each xylan concentration were used to create a dose-response graph. Using non-linear regression, the concentration that allows 50% cell viability (CC₅₀) was determined. All experiments were replicated in triplicate.

In vitro hemolytic activity

Toxicity in red blood cells was assessed by the ability of xylan to cause hemolysis in mouse erythrocytes, following the methodology outlined by Araujo et al. (2022). Erythrocytes (2% v/v) were exposed to different concentrations of xylan (500 to 7.8 µg/mL) in PBS buffer containing CaCl₂ (10 mM) for 1 h at 25 °C. After centrifugation, the supernatant was analyzed using a Benchmark Plus ELISA reader at 543 nm. Controls included PBS buffer (blank), erythrocytes without added compounds (negative control), and Triton X-100 (positive control, inducing 100% hemolysis). Commercial xylan was used as an experimental standard under the same conditions as the xylan obtained in this study. The assays were performed in triplicate. The percentage of hemolysis was determined using Equation 3.

$$\text{Hemolysis}(\mathrm{\%})=(\frac{\text{ABS sample − ABS blanc}}{\text{ABS triton X − ABS blanc}})\ast 100$$ (3)

ABS sample: Absorbance of the sample, blanc ABS: Absorbance of the negative control, ABS Triton X: Absorbance of the positive control.

In vitro immunomodulatory activity

The in vitro immunomodulatory activity assays followed the methodology proposed by Nascimento Santos et al. (2020) and Melo et al. (2022). Xylan was dissolved in distilled water and diluted to concentrations ranging from 500 to 7.8 µg/mL. Splenocyte cultures (1.0 x 10⁶ cells/mL) were exposed to xylan and incubated at 37 °C for 24 h in an incubator with 5% CO₂. After treatment, the cells were centrifuged, stained with propidium iodide and annexin V, and analyzed by flow cytometry (FACS Calibur) to assess cell viability. Cell proliferation assays were conducted under the same conditions as cytotoxicity, using CFSE.

Oxidative stress was assessed using the markers Dihydroethidium (DHE, Sigma Aldrich) to determine reactive oxygen species (ROS) levels and MitoStatus (BD Bioscience) to assess transmembrane potential. Cytosolic calcium concentration was determined using the Fluo-3AM probe (5 μM) (Thermo Fisher Scientific-USA). Immunophenotyping assays used mouse monoclonal antibodies, including anti-CD4-FITC and anti-CD8-PE for lymphocytes, anti-CD16-PE for natural killer cells, and anti-CD16/32-PercyP for monocytes (BD Biosciences). Furthermore, in the culture supernatant, the interleukins IL-2, IL-4, IL-6, IL-10, IL-17, TNF-α, and IFN-γ were determined. Nitric oxide released by cells was quantified using the Griess method.

In vitro anticoagulant activity

In vitro anticoagulant activity assays were performed using blood plasma from healthy adult mice. After centrifugation at 3,000 rpm at 4 °C for 20 min, the plasma was treated with a 3.8% sodium citrate solution (9:1) and stored at -20 °C. Coagulation tests were conducted using a semi-automatic coagulometer. The tests involved incubating 100 µL of platelet-poor plasma with different concentrations of xylan (500 – 7.8 µg/mL/20 µL) and 100 µL of APTT Pathromtin SL reagent at 37 °C for 5 min. Anticoagulant activity was assessed using activated partial thromboplastin time (APTT), thrombin (TT) and prothrombin time (PT). The negative control was performed using 0.9% sodium chloride, while heparin (7.8 µg/mL) was set as the positive control. All assays were conducted in triplicate.

In vitro antioxidant activity

The evaluation of antioxidant activity was conducted following the methodology outlined by Cruz Filho et al. (2023), with some modifications. Xylan was dissolved in distilled water at concentrations ranging from 500 to 7.8 µg/mL. The tests carried out included DPPH, ABTS, phosphomolybdenum, iron ion reduction, and hydroxyl radical capture.

The DPPH radical scavenging assay was performed using 3.9 mL of 0.06 mM DPPH methanolic solution and 0.1 mL of xylan at different concentrations ranging from 500 to 7.8 µg/mL. The system was incubated in the absence of light at a temperature of 25°C for 30 minutes. At the end of the reactions, readings were taken on a Hewlett-Packard® spectrophotometer at a wavelength of 515 nm, using methanol as the blank solution. The negative control consisted of 0.1 mL of methanol mixed with 3.9 mL of DPPH solution.

For the ABTS radical scavenging method, the ABTS⁺ cation was prepared by mixing a solution of 7 mM ABTS (10 mL) and 2.45 mM potassium persulfate (10 mL). The system was kept at room temperature (25 °C) for 16 h in the absence of light. The mixture was diluted with methanol to an absorbance of 0.7 ± 0.01 at 734 nm using a spectrophotometer (Hewlett-Packard, model 8453). In a dark environment, aliquots of 140 μL (500 to 7.8 µg/mL) of xylan were transferred to 2 mL tubes, followed by the addition of 1860 μL of ABTS reagent. Finally, the assays were incubated for 20 min. After this period, absorbances were recorded. The blank solution consisted of all reagents and methanol.

The total antioxidant activity of xylan was determined using the phosphomolybdenum assay. An aliquot of xylan (0.3 mL) in different concentrations (ranging from 500 to 7.8 µg/mL) was mixed with 3 mL of a reagent solution (28 mM sodium phosphate, 4 mM ammonium molybdate, and 0.6 M sulfuric acid). The absorbance value of the mixture was measured at 695 nm after an incubation of 90 min at 95 °C. The equipment blank consisted of 3 mL of reagent and 0.3 mL of distilled water.

The reducing power of the ferric ion is based on the reduction of Fe ³⁺ to Fe ²⁺. The tests were carried out using a volume of 2.5 mL of xylan in different concentrations (ranging from 500 to 7.8 µg/mL) added to 2.5 mL of phosphate buffer (0.2 M, pH 6.6) and 2.5 mL of potassium ferricyanide. The system was incubated at 50 °C for 20 min. At the end of the process, 2.5 mL of trichloroacetic acid was added and then centrifuged at 3000 rpm for 30 min. Finally, a 2.5 mL aliquot of the supernatant was added to 2.5 mL of distilled water and 0.5 mL of ferric chloride. The absorbances were determined using a spectrophotometer (Hewlett-Packard, model 8453) at a wavelength of 700 nm.

The hydroxyl radical capture assay was performed using an aliquot of 1.0 mL of xylan at various concentrations (ranging from 500 to 7.8 µg/mL), 0.5 mL of FeSO₄·7H₂O (1.5 mM), 0.35 mL of H₂O₂ (6 mM), and 0.15 mL of sodium salicylate (20 mM). The system was incubated in the absence of light at 37 °C for 1 h. After the incubation period, absorbances were determined using a spectrophotometer (Hewlett-Packard, model 8453) at a wavelength of 562 nm. The experimental blank used was as follows: 1.0 mL of distilled water mixed with 0.5 mL of FeSO₄·7H₂O (1.5 mM), 0.35 mL of H₂O₂ (6 mM), and 0.15 mL of sodium salicylate (20 mM).

The standards used were ascorbic acid and butylated hydroxytoluene (BHT). The concentration corresponding to 50% antioxidant activity (IC₅₀) was determined from a linear curve, obtained graphically by plotting the concentration against the percentage of antioxidant activity. All experiments were performed in triplicate.

RESULTS AND DISCUSSION

Xylan extraction yield

The xylan extraction yield was 28.44%. Different yield values are reported in the literature for extracting xylans. Cruz Filho et al. (2023) extracted xylans from the branches and leaves of Protium puncticulatum, obtaining yields of 24.32 ± 0.1% and 19.57 ± 0.3%, respectively. Gufe et al. (2023) obtained xylan from Acacia mearnsii using two extraction methods, they found that alkaline extraction assisted by ultrasound resulted in a higher yield (24%) compared to conventional alkaline extraction (21%). The variation in yield results is linked to the extraction method and the source of obtaining the xylans.

Physicochemical characterization of xylan

Analysis of monosaccharide and elemental composition

Through chromatographic analysis, only xylose and furfural were detected during the xylan characterization process being studied. Elemental analysis was conducted to determine the percentages of carbon (C), oxygen (O), and hydrogen (H) present in the xylan structure. Dinizia excelsa xylan presented the following contents: C (35.03 ± 0.1%), H (5.65 ± 0.5%), and O (59.32 ± 0.2%). Commercial xylan contains C (34.81 ± 0.3%), H (5.50 ± 0.9%), and O (59.69 ± 0.5%). No nitrogen or sulfur content was found, indicating that the isolated xylan does not contain contaminants in its structure.

These results were similar to those obtained by Cruz Filho et al. (2023). When evaluating the xylans extracted from the branches and leaves of Protium puncticulatum, the authors determined that the xylans obtained were classified as homoxylans. Furthermore, through elemental analysis, the following values were obtained: carbon 34.94 ± 0.3 and 33.79 ± 0.1%, hydrogen 6.34 ± 0.02 and 5.79 ± 0.01%, and oxygen 58.72 ± 0.1 and 60.42 ± 0.3%, respectively. These differences are associated with the source of xylan extraction.

Fourier transform infrared spectroscopy (FTIR) analysis

The functional groups present in the xylan structure were determined by FTIR. Figure 1a, b presents the FTIR spectra of xylan extracted from Dinizia excelsa wood and commercial xylan. The spectral bands characterizing different xylans were previously identified by Liu et al. (2021), Cruz Filho et al. (2023) and Wen et al. (2024).

Figure 1
FTIR spectra of wood xylan from Dinizia excelsa (a) and commercial xylan (b) respectively.

The results showed that the FTIR spectrum of Dinizia excelsa xylan was similar to commercial xylan. The bands at 3490 and 3496 cm^-1 were attributed to the stretching vibration of the hydroxyl group (-OH). The signals at 2976 and 2972 cm^-1 correspond to the symmetric stretching of the methyl group (CH). The peaks at 1643 and 1640 cm^-1 were attributed to the H-O-H angular deformation characteristic of water. The bands at 1420 and 1463 cm^-1 correspond to the angular deformation of CH₂. The bands at 1334 and 1363 cm^-1 were attributed to CH bending vibration. The region between 1200 and 1000 cm^-1 is attributed to the stretching vibrations of the -OH side group and the glycosidic C-O-C vibrations. The presence of the β-1,4 bond was confirmed by the bands at 1061 and 1047 cm^-1, respectively. Finally, the bands at 895 and 899 cm^-1 were attributed to the stretching and deformation of the C-O-C, C-C-O, and C-CH bonds present in the β-glycosidic bonds between the xylanopyranoses of the main chain.

Magnetic Resonance Analysis (2D MRI): Heteronuclear Single Quantum Coherence (HSQC)

2D NMR analyses are more efficient for elucidating xylans because the spectra provide more detailed structural information with less ambiguity compared to 1D NMR analyses (Giummarella et al. 2019). The analysis performed was 2D HSQC NMR, this technique is based on correlations between hydrogens and directly bonded carbons (Petersen et al. 2014).

The HSQC spectra of Dinizia excelsa wood and commercial xylan were presented in Figures 2a, b respectively. The signals were previously assigned by Sharma et al. (2020) and Cruz Filho et al. (2023).

The spectra (Figure 2a, b) showed similarities, presenting the following 13C/1H correlations: 101.6/4.29 corresponding to C1/H1 in β-D-xylopyranoside (X1), 76.2/3.20 attributed to C3/H3 in β-D-xylopyranoside (reducing terminal unit), 73.2/3.55 C3/H3 in 4-O-methylglucuronic acid (U3), 71.8/3.20 related to C2/H2 in 4-O-methylglucuronic acid, 70.2/3.64 attributed to C3/H3 in α-D-xylopyranoside (reducing terminal unit), 65.5/3.70 attributed to C5/H5 in β-D-xylopyranoside (non-reducing terminal unit), and finally, 60.4/3.51 referring to the C/H of methoxyl in 4-O-methylglucuronic acid, respectively.

Figure 2
HSQC spectra of wood xylan from Dinizia excelsa (a) and a commercial sample (b).

Determination of molecular mass by gel permeation chromatography (GPC)

The molecular weight of xylans was determined using gel permeation chromatography. The chromatograms are shown in Supplementary Material - Figures S1, S2. From this analysis, it was possible to determine the values of Mw (weighted average molecular mass), Mn (numerical average molecular mass), and Mw/Mn (polydispersity), respectively. The Mw, Mn, and Mw/Mn values for xylan from Dinizia excelsa wood were 38.32 g/mol, 24.91 g/mol, and 1.53. For commercial xylan: 37.93 g/mol, 23.87 g/mol, and 1.58 g/mol, respectively. These values are similar to those reported by Cruz Filho et al. (2023) for the evaluation of xylans from the branches and leaves of Protium puncticulatum, obtained Mn, Mw, and Mw/Mn values of 25.10 g/mol, 35.89 g/mol, and 1.42 for the branches, and 24.89 g/mol, 35.74 g/mol, and 1.4 for the leaves, respectively. These results indicate that the xylan obtained from Dinizia excelsa has a low molecular.

Cytotoxicity assays

Assessing toxicity is a crucial step in determining the nutritional or biomedical potential of a compound. Cytotoxicity curves are shown in Figures S3a-d. The CC₅₀ values for xylan obtained from Dinizia excelsa ranged from 249 µg/mL to >500 µg/mL, indicating higher cytotoxicity for J774 macrophage lines (CC₅₀ 249 ± 0.1 µg/mL) and lower cytotoxicity for Vero cells (CC₅₀ >500 µg/mL). In relation to the HepG2 lineage, it exhibited a CC₅₀ of 335.1 ± 1.2 µg/mL, while the V79 fibroblasts showed a value of 416.6 ± 0.9 µg/mL. The CC₅₀ values demonstrate that the xylan obtained has low cytotoxicity. The percentage of hemolysis was below 10%, characterizing xylan as non-hemolytic (Figure S4). The results were close to those obtained for commercial xylan, with CC₅₀ varying from 250.1 µg/mL to > 500 µg/mL. In terms of cytotoxicity, the order is as follows: J774 macrophages (250.1 ± 0.9 µg/mL), HepG2 (336 ± 3.0 µg/mL), V79 fibroblasts (417 ± 2.1 µg/mL), and Vero cells (> 500 µg/mL) (Figure S3a-d). Furthermore, it presented a hemolytic activity percentage lower than 10% (Figure S4).

The literature describes xylan as a polysaccharide with low cytotoxicity. Wang et al. (2023) studied xylan nanocrystals obtained from biological waste and found CC₅₀ values >200 µg/mL for HL-7702 hepatocytes and A549 lung epithelial cells. Gutiérrez-Hernandez et al. (2023) formulated a xylan-based hydrogel and demonstrated that the gel can enhance the cellular proliferation of osteoblasts, indicating that xylan is non-toxic to bone connective tissue. In a similar way, xylan extracted from sawdust, as studied by Sharma et al. (2020), exhibited cell viability of over 50% at a concentration of 200 µg/mL for HT29 human adenocarcinoma cells and L929 fibroblasts. The CC₅₀ values found in this study enabled the classification of the xylan extracted from Dinizia excelsa as having low toxicity in the experiments evaluated.

In vitro immunomodulatory activity

Xylans are described in the literature as potential immunomodulatory agents. The results of the immunomodulatory activity tests of xylan extracted from Dinizia excelsa and commercial xylan were presented in Supplementary Material - Tables SI and SII. The results indicated that the xylans were not cytotoxic, as they demonstrated cell viability above 95%. Furthermore, it was observed that xylan stimulated cell proliferation. Similar results were obtained by Shi et al. (2014) and Cruz Filho et al. (2023). The authors found that the xylans evaluated in their studies were capable of inducing the proliferation of immune cells, in addition to having low toxicity.

Cytokine analysis revealed that xylans induced the production of anti-inflammatory cytokines (IL-4, IL-10) and IL-6, while others (IL-2, IL-17, TNF-α, IFN-γ) remained at basal levels. The anti-inflammatory profile was confirmed by the reduction in nitric oxide production. These results were similar to those obtained by Anindya et al. (2019) when evaluating xylan from pineapple stem residues. The authors found that this polysaccharide has the potential to act as an anti-inflammatory agent. However, it was different when compared to those obtained by Cruz Filho et al. (2023), who found a predominant pro-inflammatory response to xylans extracted from Amazonian plants.

The immunomodulation mechanism for xylans is not fully elucidated; however, it is known that immune cells can recognize these polysaccharides and induce a specific response (Shi et al. 2014, Zhang et al. 2021). It is worth noting that the immunological response is influenced by the chemical structure of xylans; different structures can induce varying responses (Shi et al. 2014, Tiwari et al. 2020).

In relation to oxidative stress, xylan increased the production of reactive oxygen species, cytosolic calcium levels, and mitochondrial membrane potential, indicating cellular activation without causing cell death. In immunophenotyping assays, xylans induced an increase in the population of CD8+ lymphocytes, suggesting their potential as an immunomodulatory agent in vitro. These findings indicate that the xylan studied is a promising immunomodulatory agent.

In vitro anticoagulant activity

Six solutions of different concentrations of xylan were analyzed using the Prothrombin Activation Time (PT), Partial Thromboplastin Activation Time (APTT) and Thrombin Time (TT) tests. These tests were selected because they are the primary assessments conducted in scientific research and laboratory routines to monitor anticoagulant activity. The results of anticoagulant activity were presented in Table SIII. These results show that the evaluated xylan has low anticoagulant activity compared to heparin. Similar results were obtained by Cruz Filho et al. (2023), who found that xylans from Amazonian plants exhibited low anticoagulant activity. However, modifications in the xylan structure can induce increased activity. Chen et al. (2020) and Cheng et al. (2017) found that sulfated xylans exhibited superior results in anticoagulant activity.

In vitro antioxidant activity

Xylans are described in the literature as polysaccharides with promising antioxidant activity. Table SIV presents the results of the in vitro antioxidant activity of xylans compared to the standards ascorbic acid and butylated hydroxytoluene (BHT). The results presented in Table SIV show that xylans in various assays were able to promote antioxidant activity ranging from 4 to 47 %. Furthermore, it did not demonstrate a concentration sufficient to induce 50% antioxidant activity (IC₅₀). These values were lower when compared to the standards of ascorbic acid and butylated hydroxytoluene (BHT). Based on the results, xylan from Dinizia excelsa was considered a weak antioxidant agent under the experimental conditions evaluated.

CONCLUSIONS

Based on the topics discussed, it can be concluded that the xylan obtained is a homoxylan, characterized by its non-toxicity towards various normal cells and the absence of significant hemolytic effects. Furthermore, its low anticoagulant activity suggests a favorable safety profile. The outstanding immunomodulatory activity positions it as a potential anti-inflammatory agent, offering benefits in regulating the immune system. On the other hand, despite its low antioxidant activity, the therapeutic attributes of this substance as an anti-inflammatory and immunomodulatory agent still make it a promising candidate for further investigations and potential clinical applications.

SUPPLEMENTARY MATERIAL

Figures S1-S4.

Tables SI-SIV.

ACKNOWLEDGMENTS

The study was funded by Fundação de Amparo à Ciência e Tecnologia de Pernambuco (FACEPE), Process - FACE-04.03 / 19 - Researcher Research Grant - Process BFP-0038-0 and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Process - 306865 / 2020-3.

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Publication Dates

• Publication in this collection
  29 Nov 2024
• Date of issue
  2024

History

• Received
  12 Mar 2024
• Accepted
  17 Aug 2024