Abstract

Introduction: Radiation has adverse effects on brain tissue. Natural products have anti-inflammatory and anti-inflammatory properties. This study investigates the protective effect of alginate extract from matcha against gamma radiation damage.

Methods: We isolated and characterized arabinogalactans (AG) from matcha powder. We randomly divided thirty-two male rats into four equal groups (n = 8 rats/group). The first group served as the control, while the second group administered MAG orally to the rats for 14 days. The third group of rats underwent a single dose of 10 Gy of head-localized irradiation (Rad). The fourth group, rats, received MAG for 7 days before and after 10 Gy γ irradiation (B &A). We examined the brain for markers of oxidative stress and inflammation. We used immunohistochemistry to monitor tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1β), interleukin 6 (IL-6), and apoptosis-regulating proteins BAX and BCL2. We measured levels of glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) in brain tissues as indicators of oxidative stress. Western blot analysis assessed apoptotic markers like cleaved caspase-3 and neurotrophic factors, including Brain-derived neurotrophic factor (BDNF). Additionally, Double Delta Ct QPCR was employed to evaluate gene expression changes in monoamine oxidase (MOA), acetylcholinesterase (AChE), and nuclear factor kappa B (NF-κB). Histopathological tests were performed to verify the effects of therapy, elucidating neuronal damage and inflammation.

Results: Radiation exposure elevated oxidative stress indicators, pro-inflammatory cytokines, and apoptotic proteins, while diminishing antioxidant and BDNF levels. The MAG therapy (B & A) decreased MDA levels and pro-inflammatory cytokines (TNF-α, IL-1β). MAG lowers SOD and GSH levels while enhancing catalase activity. It also altered the concentrations of apoptosis-related proteins such as BAX, BCL2, and cleaved caspase-3. QPCR results indicated that MAG reinstated the modified MAO-A, AChE, and NF-κB genes. Histological analyses validated the protective properties of MAG, demonstrating decreased tissue injury and inflammation.

Conclusion: This study demonstrates that matcha-derived arabinogalactans may protect neurons from radiation-induced cerebral injury. This is likely attributable to their antioxidant, anti-inflammatory, and anti-apoptotic characteristics.

Introduction

In recent years, the potential health benefits of natural plant extracts have received increasing attention in medical research. Among these compounds, arabinogalactans, a polysaccharide abundant in plant species, seem to have promising therapeutic effects1. The finely ground powder of mature and processed tea leaves is particularly recognized for its high content of bioactive compounds, including matcha arabinogalactans, making it important to investigate potential radiosafety compounds2, 3.

Gamma rays, an ionizing radiation, are widely used in medical research and cancer treatment. However, exposure to gamma rays carries a high risk of oxidative stress, inflammation, and damage to biological tissues, especially the brain4, 5. The central nervous system is extremely sensitive to radiation damage due to its complex cellular structure and metabolic demands.

Proteoglycans, or arabinogalactans, are a class of polymers associated with plant growth and development. They are found in many tissues of higher plants, such as the outer cell matrix, cell walls, and plasma membranes, and the larch tree (Larix sp.) is considered the most abundant source in the USA6. Larch-derived arabinogalactan is recognized as a source of dietary fiber, but it is also believed to have potential therapeutic value as a stimulant in immune and cancer programs7. The molecular weight of arabinogalactans typically ranges from 10 kDa to 4000 kDa. Their protein content is usually less than 10%, consisting mainly of proline/hydroxyproline, alanine, serine, and threonine. More than 90% of arabinogalactans are polysaccharides, primarily β-(1-3)-galactan chains with a β-(1-6)-galactosyl side chain, usually terminated by an arabinyl residue8. The structural investigation of arabinogalactan-proteins, which are crucial signaling molecules of the plant cell wall, has been widely studied in angiosperms. However, there is a scarcity of information on gymnosperms9. Polyphenolic compounds have been shown to affect pro-inflammatory signaling, pro-inflammatory cytokines, and radiation-induced damage. Anti-inflammatory properties of certain compounds, such as anthocyanin, curcumin, and resveratrol, have also been identified as significant radioprotectors10, 11. Arabinogalactans possess antioxidant, anti-inflammatory, and immunomodulatory activities that can mitigate the detrimental effects of radiation on cerebral tissue. Moreover, scaffold-derived arabinogalactans offer the advantage of being a simple natural source without adverse effects12.

Our goal in this study was to investigate the neuroprotective potential of arabinogalactans extracted from matcha against gamma radiation-induced brain damage. Rats are an established model for studying the physiological and pathological effects of radiation on brain function. Using a mouse model, we can better mimic the response to human brain exposure to gamma radiation.

Methods AG Extraction and Characterization

Matcha, a powdered green tea, was graciously provided by Hangzhou Shandi Tea Industry Co., Ltd. (Hangzhou, China). The AG was obtained using the Bartle13 approach. The dried plant material was subjected to a 24-hour aqueous extraction process at a ratio of 1:10 (w/v) with continuous shaking at 4 °C. The solid plant material was then extracted and heated at 92 °C for 10 minutes. The extract was held overnight at 4 °C to facilitate the precipitation of proteins while ensuring that the AG remained soluble. The proteins were removed by centrifugation at 15,000 g and 4 °C for 10 minutes. To separate a high molecular weight (HMW) fraction rich in polysaccharides and AG proteins, the extract was mixed with cold 100% ethanol until it reached an 83% (v/v) concentration. The precipitate was then subjected to centrifugation at 15,000 g and 4 °C for 20 minutes. The HMW fraction was freeze-dried. The AGP was isolated from the HMW fraction through selective precipitation using β-glucosyl-Yariv reagent (βGlcY), as described by Classen et al.14 and Paulsen et al. 15.

High-Performance Liquid Chromatography (HPLC) Analysis

HPLC analysis was performed using an Agilent 1260 series system equipped with a Zorbax Eclipse Plus C8 column (4.6 mm × 250 mm, 5 µm particle size). The mobile phase consisted of two components: A (water) and B (0.05% trifluoroacetic acid in acetonitrile). The separation was carried out using a linear gradient elution at a flow rate of 0.9 mL/min as follows: 0–1 min: 82% A, 1–11 min: 75% A, 11–18 min: 60% A, 18–22 min: 82% A, and 22–24 min: 82% A (re-equilibration phase).

The column temperature was maintained at 40 °C. Detection was performed at a wavelength of 280 nm using a multi-wavelength detector. The injection volume for each sample was 5 µL. Standards of gallic acid and chlorogenic acid were prepared at known concentrations to generate calibration curves. Sample solutions were then analyzed under identical conditions, and their concentrations were determined by interpolating peak areas into the standard calibration curves.

Fourier Transform Infrared (FTIR) Spectroscopy

FTIR analysis was performed to identify the functional groups present in the extracted arabinogalactans. The spectra were recorded using a Bruker VERTEX 80 FTIR spectrometer equipped with a Platinum Diamond ATR accessory. The analysis was conducted in the range of 4000–400 cm⁻¹ with a resolution of 4 cm⁻¹. The sample was placed directly on the ATR crystal, ensuring full contact, and spectra were obtained after 16 scans to enhance the signal-to-noise ratio.

Assessment of total phenolic content

Zilic et al.16 assessed the total phenolic content using the Folin-Ciocalteu method. The extract (100 µL) was placed in a test tube, and distilled water was added to make it 3.5 mL. Next, 250 µL of Folin-Ciocalteu’s reagent was added to start the oxidation process. We added 1.25 mL of 20% aqueous sodium carbonate (Na2CO3) solution to the mixture after the reaction to stop it. After waiting for 40 minutes, we measured the absorbance at 725 nm, using the blank as a reference. We quantified the total phenolic content from the calibration curve using gallic acid and expressed the results in milligrams (mg GAE) per gram of the sample.

Determination of total flavonoid content

The total flavonoid content was determined using the aluminum chloride (AlCl3) colorimetric method as outlined by Zilic et al. 17. In summary, 300 µL of 5% sodium nitrite (NaNO2) was mixed with 100 µL of extract. After 6 minutes, 300 µL of a 10% AlCl3 solution was added, and the total volume was adjusted to 2.5 mL with distilled water. After 7 minutes, 1.5 mL of 1 M NaOH was added, followed by centrifugation at 5000 g for 10 minutes. The absorbance of the supernatant was measured at 510 nm for the solvent blank. The total flavonoid concentration was determined using a calibration curve derived from catechin and represented as μg CE/g sample.

Radical DPPH scavenging efficacy

Using the stable 1,1-Diphenyl-2-picryl-hydrazyl (DPPH•), we evaluated the capacity to scavenge free radicals. For this ATR-FTIR study, we used a Bruker VERTEX 80 (Germany) coupled with a Platinum Diamond ATR. A diamond disk with a refractive index of 2.4 and a resolution of 4 cm−1 acts as an internal reflector in this device, operating in the 4000–400 cm−1 range.

The overall reaction volume was 4.0 mL, and the final concentration of DPPH• was 50 μM. After 60 minutes, we measured the absorbance at 517 nm (A) in comparison to a pure methanol blank. The following equation was used to obtain the % inhibition of the DPPH free radical:

Calculation of inhibition (%) = 100 × (Acontrol − Asample) / Acontrol.

The antioxidant activity was measured in micrograms of Trolox equivalent (TE) per gram of material, according to a calibration curve set up with Trolox.

Assessment of ABTS radical scavenging activity

The ABTS radical scavenging activity was evaluated by making stock solutions of the reagent according to the method described by Hwang and Do Thi18. This involved mixing 7 mM of aqueous ABTS* with 2.45 mM of potassium persulfate in equal volumes, then incubating the mixture for 16 hours at room temperature (25 °C) in the dark. To create a working solution, 1 mL of ABTS* solution was mixed with 60 mL of a 50:50 (v/v) ethanol and water mixture. This mixture was tested using a Spectro UV-VIS Double Beam spectrophotometer (Model UVD-3500, Labomed, Inc., La Cienega Blvd., Los Angeles, CA 90034, U.S.A.) to ensure an absorbance of 1.0 ± 0.02 units at 734 nm. For one hour in the dark, 50 µL of the extracts were allowed to react with 4.95 mL of the ABTS* solution. Using a spectrophotometer, the absorbance was determined at a wavelength of 734 nm. A standard curve was created using Trolox. Data were shown as micrograms of Trolox equivalents (TE) per gram.

Determination of LD50 of MAG in rats

In drug screening, the measurement of LD50 (the dosage lethal to 50% of the tested animal population) is typically a first step in evaluating the hazardous properties of a chemical. This initial toxicity evaluation offers insights into potential health risks associated with short-term drug exposure and is one of the initial screening tests conducted on all substances19, 20. The examined substances were delivered in incremental dosages, and the LD50 was ascertained21. We estimated the optimal dose for assessing the in vivo antitumor efficacy of the examined drugs to be one-tenth of their LD50 value.

Gamma Irradiation

A Canadian gamma cell 40 (137Cs) at the National Center for Radiation Research and Technology (NCRRT) in Cairo, Egypt was used to irradiate the rats' head only exposure at dose 10 Gy, while the rest of the body was shielded with 5-mm-thick lead. The dose rate was 0.585 rad/sec. Rats were anesthetized via intraperitoneal (IP) injection of sodium pentobarbital (50 mg/kg) to maintain immobility during the irradiation period. The animals were irradiated individually rather than concurrently. Each rat was separately anesthetized, positioned in the gamma cell, and exposed to the radiation source under controlled conditions.

Animal Experiments

We recruited mature male albino rats ranging in weight from 180 to 200 g from the NCRRT's animal house in Cairo, Egypt. We fed the animals a regular pellet diet and provided water ad libitum. We kept them in a controlled environment with a 12-hour light-dark cycle, at room temperature and pressure.

Experimental Groups and Treatment Protocol

A total of 32 male Wistar albino rats, weighing between 180 and 200 grams and aged 6 to 8 weeks, were randomly divided into four groups (n = 8):

Group 1 (Control): Rats received an oral gavage of 0.9% sodium chloride solution (saline) daily for 14 days. Group 2 (MAG): Rats received oral gavage of Matcha-derived arabinogalactans (MAG) at a therapeutic dose daily for 14 days. Group 3 (Localized Irradiation): Rats were exposed to 10 Gy gamma radiation targeted to the head using a precise collimator to minimize radiation to other body regions22. Group 4 (Pre-MAG + Radiation + Post-MAG): Rats received MAG at the therapeutic dose for 7 consecutive days prior to 10 Gy head-localized gamma radiation, followed by continued administration of MAG for 7 days post-irradiation.

On day 15, animals were anesthetized using intraperitoneal injection of sodium pentobarbital (60 mg/kg) to ensure minimal stress. Blood samples were collected via cardiac puncture. Following blood collection on day 15, animals were humanely euthanized using an overdose of sodium pentobarbital (150 mg/kg) administered intraperitoneally, followed by cervical dislocation to ensure death.

The study was conducted following approval by the Institutional Animal Care and Use Committee (IACUC) of the National Centre for Radiation Research and Technology (NCRRT), Cairo, Egypt, under approval number (permission No P/35A/24) on May 2, 2024. All procedures complied with the ethical guidelines outlined by the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals.

Protein extraction and purification

Rats were sacrificed, and brain tissues were dissected to prepare 10% tissue homogenates in ice-cold phosphate buffer for analysis of oxidative stress and inflammation markers. The TriFast technique (Peqlab, VWR) was used for protein extraction, allowing the simultaneous isolation of RNA, DNA, and protein. Tissue samples (50–100 mg) were homogenized in 1 mL of TriFast using either a Glass-Teflon or power homogenizer, ensuring the sample volume did not exceed 10% of the TriFast solution. After the addition of 0.2 mL of chloroform per 1 mL of TriFast, the mixture was vigorously shaken for 15 seconds and incubated at room temperature for 3–10 minutes. The mixture was then centrifuged at 12,000 × g for 5 minutes. The protein-containing phase was separated and precipitated with 0.5 mL of isopropanol per 1 mL of TriFast, incubated on ice, and centrifuged at 12,000 × g for 10 minutes at 4°C.

The precipitated protein pellet was washed three times with 0.3 M guanidinium hydrochloride in 95% ethanol, incubated at room temperature for 20 minutes, and centrifuged at 7,500 × g for 5 minutes. The pellet was then mixed with 2 mL of 100% ethanol, incubated for 20 minutes, centrifuged, vacuum-dried, and reconstituted in buffer. Residual insoluble material was removed by additional centrifugation at 10,000 × g for 10 minutes at 4°C. The protein-containing solution was stored at −20°C.

Protein Quantification

Protein concentration was determined using the Bradford method. Each sample (50 µL) was mixed with 50 µL of distilled water and 200 µL of Coomassie Brilliant Blue G-250 reagent. After a 5-minute incubation, absorbance was measured at 595 nm. Protein concentrations were calculated using a BSA standard curve prepared under the same conditions.

Gel Electrophoresis

Acrylamide-bisacrylamide gels were prepared with 12% resolving and 4% stacking layers, following the method of Bamdad et al. Protein samples (30 µg) were loaded onto the gel and separated at 75 V for the stacking layer and 125 V for the resolving layer over 2 hours. The gel was stained with 0.1% Coomassie Blue R-250 in a methanol-acetic acid solution (1:3:6) and destained to visualize the protein bands. Protein bands were visualized using the Geldoc-it system (UVP, England) and analyzed with TotalLab software to determine molecular weight and protein expression.

Molecular Assessments

We prepared blotting buffers for the blotting technique, which consist of 25 mM Tris, pH 7.4, 0.15 M NaCl, and 0.1% Tween 20. We prepared a blocking solution using 2–5% nonfat dry milk, adjusted to a pH of 7.4. We produced an antibody solution in the blotting buffer using dried milk (1–5% fat), then adjusted the solution according to the proteins separated by SDS-PAGE. We transferred these proteins onto HybondTM nylon membranes (GE Healthcare). β-actin was used as the control protein and was incubated at ambient temperature in the blocking solution for one hour. Next, the membrane was placed in a solution containing an anti-BDNF antibody [EPR1292, Abcam, USA, 15 kDa] and left at 4 °C overnight.

QPCR Analysis Using Double Delta Ct MAO-A gene expression

TRIzol Reagent (15596026, Life Technologies, USA) was used for total RNA purification from blood samples according to the manufacturer’s protocol. TRIzol Reagent’s unique combination of phenol, guanidine isothiocyanate, and other components enables the sequential precipitation of RNA, DNA, and proteins from a single sample. After homogenization with TRIzol, chloroform was added to separate the solution into three layers: an aqueous RNA layer, an interphase, and a red organic layer containing DNA and proteins. RNA was then precipitated with isopropanol. DNA was separated from the organic/interphase layer with ethanol, and protein was obtained by isopropanol precipitation.

For RNA extraction, the sample was transferred to a centrifuge tube and treated with RBC Lysis Buffer, then centrifuged. The pellet was washed and resuspended in DPBS, then centrifuged again. TRIzol reagent was added, and the sample was stored at −80°C. After incubation, chloroform was added, and the mixture was centrifuged at 12,000 × g at 4°C for 15 minutes. RNA in the aqueous layer was precipitated with isopropanol, washed with 75% ethanol, air-dried, and resuspended in RNase-free water or buffer.

The QuantiTect Reverse Transcription Kit was employed to convert RNA into cDNA, ensuring removal of genomic DNA contamination. The material was incubated at 42°C for reverse transcription, followed by 95°C, and the cDNA was used immediately for PCR or stored at −20°C.

Preparation of quantitative PCR reaction

The Rotor-Gene Q (Qiagen, USA) was utilized to conduct real-time PCR for the evaluation of mRNA levels of AChE and MAO-A genes. β-actin was used as the housekeeping gene. Amplifications were carried out using the Maxima SYBR Green/Fluorescein qPCR Master Mix (2X), which contains SYBR Green I dye, fluorescein passive reference dye, and dUTP for optional carryover contamination control. After thawing and mixing, the required components were added to make a reaction master mix for each 25 μL reaction. Template DNA (≤100 ng/reaction) was added, and the thermal cycler was programmed for an initial heating step of 95°C for 10 minutes, followed by 45 cycles of 95°C for 10 seconds, 60°C for 15 seconds, and 72°C for 15 seconds. Melting curve analysis was performed between 72°C and 95°C at a rate of 1°C per second.

Table 1.

Primer primers for genes under study

Genes Primers References AChE 5′-AAT CGA GTT CAT CTT TGG GCT CCC CC-3′ Xia et al. 2022 5′-CCA GTG CAC CAT GTA GGA GCT CCA-3′ GAPDH 5′-AAC GGA TTT GGC CGT ATT GG-3′ Xia et al. 2022 5′-CTT CCC GTT CAG CTC TGG G-3′ MAO-A 5’-GCCAAGATTCACTTCAGACCAGAG-3’ Alkhawaldeh and Bardaweel, 2023 5’-TGCTCCTCACACCAGTTCTTCTC-3’

One microgram of total RNA was converted into single-stranded complementary DNA (cDNA) using the QuantiTect Reverse Transcription Kit (Qiagen, USA). The gDNA Wipeout solution was used to remove genomic DNA. Random hexamers served as primers. Amplification began with 30 ng of total cDNA and ended with 300 nM of each primer pair (Table 1). Each sample underwent triplicate real-time PCR, and the average results were used for further analysis. Ubiquitin 5 (UBQ5) served as the reference gene. Data were generated automatically based on threshold Cycle (Ct) values. The ΔCt values for each sample were calculated by adjusting to the average Ct value of the reference genes.

Following Livak and Schmittgen23, the relative expression of the target gene was determined using the 2-ΔΔCt method. The control group served as the calibrator, and the other dietary groups served as experiments. The fold change was calculated as (2-ΔΔCt).

Detection of cytokines

Commercially available kits from R&D Systems, USA, were used to quantify the levels of cytokines in rat blood, including Interleukin-6 (IL-6), Interleukin-1 beta (IL-1β), and Tumor Necrosis Factor-alpha (TNF-α). The manufacturer's instructions were followed precisely. The standard cytokine curve was on the same sample plate as the measured cytokine concentrations.

Immunohistochemistry for BAX and BCL2

Rat brain tissue was either freshly collected or obtained from a previously fixed specimen. For fresh tissue, brains were immersed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 4 hours at 4°C. For frozen sections, tissues were fixed in 4% paraformaldehyde overnight, then cryoprotected in 30% sucrose in PBS until they sank, typically overnight at 4°C. The tissue was embedded in OCT compound, frozen using liquid nitrogen, and sectioned (10–20 µm thick) using a cryostat. Sections were mounted onto glass slides.

Sections were incubated in blocking solution containing 5% bovine serum albumin (BSA) in PBS for 1 hour at room temperature to prevent non-specific binding and then treated with 0.1% Triton X-100 in PBS for 15 minutes at room temperature for permeabilization. Antigen retrieval was performed by heating sections in citrate buffer (pH 6.0) at 95°C for 20 minutes, followed by cooling and washing. Sections were incubated overnight at 4°C with primary antibodies against Bax (Rabbit anti-Bax, diluted 1:200) and Bcl-2 (Rabbit anti-Bcl-2, diluted 1:200).

After washing three times with PBS, the sections were incubated with appropriate secondary antibodies conjugated to fluorophores for 1–2 hours at room temperature in the dark. The sections were washed again, and nuclei were counterstained with DAPI (1 µg/mL) for 5 minutes. Finally, the sections were mounted in an anti-fade medium and examined under a fluorescence microscope. Images were captured with filters corresponding to the specific fluorophores, and Bax and Bcl-2 expression patterns were analyzed both qualitatively and quantitatively.

Oxidative Stress Parameters

Malondialdehyde (MDA) levels (a marker of lipid peroxidation indicating oxidative injury), superoxide dismutase (SOD) activity (a key antioxidant enzyme), reduced glutathione (GSH) levels (a major cellular antioxidant), catalase activity (an antioxidant enzyme critical for hydrogen peroxide clearance), and nuclear factor Kappa-B (NFκB) (the key transcriptional regulator of the inflammatory pathway) will be determined in the brain tissues.

Western blot analysis will be employed to assess the expression levels of apoptosis markers (cleaved caspase-3) and neurotrophic factors (brain-derived neurotrophic factor, BDNF). In addition, amyloid precursor protein (APP) levels will be analyzed by western blotting.

Histopathological analysis

We preserved skeletal muscle tissue samples in 10% formol saline, then sectioned, cleaned, and dehydrated them in increasing concentrations of alcohol. We cleared the dehydrated specimens in xylene, fixed them in paraffin blocks, and sectioned them to a thickness of 4–6 µm. We removed the paraffin from the tissue slices using xylene and then stained them with hematoxylin and eosin (H&E). An electric light microscope was used for histopathological analysis24.

Statistical analysis

The sample size for this study was determined according to the 3Rs ethical principles (replacement, reduction, and refinement) in animal research. A post hoc power analysis was conducted using G*Power (version 3.1.9.7) to assess the sufficiency of the sample size25. Based on an effect size (d = 0.7), an alpha level (α) of 0.05, 80% power, and type II error, the analysis indicated that a total sample size of 32 rats (8 rats per group) was sufficient for detecting statistically significant differences. Values for all parameters are presented as the mean ± SEM. To determine if there were statistically significant differences among the groups, we used SPSS 23.0 and a one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison tests. We considered findings significant if p

× Figure 1 . Characterization of MAG extract . a ) Attenuated Total Reflectance – Fourier Transform Infrared Spectroscopy (ATR-FTIR) spectrum of MAG, illustrating key peaks corresponding to functional groups, including O-H (3284 cm⁻¹), C-H (2921 cm⁻¹), C=O (1737 cm⁻¹), and C-O-C (1075 cm⁻¹), confirming the polysaccharide and phenolic composition of the extract, b ) Standard calibration curve for total phenolic content using Gallic acid as a reference, showing a linear relationship between absorbance and concentration (R² > 0.99), c ) Standard calibration curve for total flavonoid content using catechin as a reference, demonstrating a linear correlation (R² > 0.99), d ) DPPH radical scavenging assay calibration curve using Trolox as a standard (R² > 0.99), quantifying antioxidant capacity, e ) ABTS assay calibration curve using Trolox as a reference (R² > 0.99), illustrating the extract's antioxidant potential. Figure 1 . Characterization of MAG extract . a ) Attenuated Total Reflectance – Fourier Transform Infrared Spectroscopy (ATR-FTIR) spectrum of MAG, illustrating key peaks corresponding to functional groups, including O-H (3284 cm⁻¹), C-H (2921 cm⁻¹), C=O (1737 cm⁻¹), and C-O-C (1075 cm⁻¹), confirming the polysaccharide and phenolic composition of the extract, b ) Standard calibration curve for total phenolic content using Gallic acid as a reference, showing a linear relationship between absorbance and concentration (R² > 0.99), c ) Standard calibration curve for total flavonoid content using catechin as a reference, demonstrating a linear correlation (R² > 0.99), d ) DPPH radical scavenging assay calibration curve using Trolox as a standard (R² > 0.99), quantifying antioxidant capacity, e ) ABTS assay calibration curve using Trolox as a reference (R² > 0.99), illustrating the extract's antioxidant potential. Results FTIR analysis

We used Fourier transform infrared spectroscopy (FTIR) to identify the functional groups of arabinogalactans isolated from the scaffolds (Figure 1a). The FTIR spectrum revealed several absorption bands indicating different functional groups. The broadening of the 3242.40 cm⁻¹ peak indicates O-H broadening vibrations, which are associated with hydroxyl groups. Phenolic compounds and polysaccharides commonly contain these groups. The peak at 2921.27 cm⁻¹ coincides with C-H stretching vibrations, indicating the presence of aliphatic chains in the extract. The peak at 1739.94 cm⁻¹ is caused by C=O stretching vibrations, which are common in carbonyl groups such as esters or carboxylic acids. The band at 1638.21 cm⁻¹ is caused by C=C stretching vibrations, which are likely related to the aromatic ring of phenolic compounds. Moreover, there is a broad peak around 3300–3500 cm⁻¹, which could correspond to O-H stretching (typical for gallic acid) or N-H stretching (typical for melatonin). There is a strong peak around 1700 cm⁻¹ (specifically at 1713 cm⁻¹), which could be the C=O stretching of carboxylic acid in gallic acid or the amide C=O in melatonin. The 1414.24 cm⁻¹ peak corresponds to C-H bending vibrations, common in various organic compounds. The band at 1243.67 cm⁻¹ suggests the presence of ether or ester functional groups (C-O propagation vibration). The distinguishing characteristics of glycosidic bonds in polysaccharides, which manifest as a strong band at 1078.56 cm⁻¹, are the C-O-C propagation vibrations (1017.02 cm⁻¹, 880.95 cm⁻¹, 779.34 cm⁻¹, 616.59 cm⁻¹, 579.45 cm⁻¹).

Total phenolic, flavonoid contents, and antioxidants

The total phenolic content of the extract was found to be 341.74 μg GAE/g (Figure 1b). This indicates a high content of phenolic compounds known to have antioxidant properties. We also measured the total flavonoid content, which was 95.00 μg CE/g. Flavonoids are important for preventing oxidative stress. The total flavonoid content was determined by means of a calibration curve prepared with catechins (Figure 1c) and expressed as micrograms of catechin equivalent (μg CE) per g. In our study, we tested the antioxidant properties of arabinogalactans extract using two different assays: DPPH and ABTS. The DPPH assay (Figure 1d) showed an antioxidant activity of 317.43 µg TE/g, while the ABTS assay showed a slightly higher activity of 352.96 µg TE/g. These results indicate that the scaffold has strong antioxidant properties for the production of arabinogalactans water from it. If the observed absorbance value exceeded the linear range of the standard curve, further dilution was performed. The ABTS standard curve was generated utilizing Trolox (Figure 1e). Results were presented as μg Trolox equivalents (TE) per gram.

× Figure 2 . The Brain-Derived Neurotrophic Factor (BDNF) before and after treatments . a ) Western blot of BDNF expression, with molecular weight markers (250–10 kDa) shown on the left. β-actin (~42 kDa) was used as a loading control for normalization, b ) Computerized analysis of Brain-Derived Neurotrophic Factor (BDNF) antibody protein expression level for samples, c ) Densitometric analysis of BDNF expression across experimental groups under different conditions, showing anti- Brain-Derived Neurotrophic Factor (BDNF) band calculation for samples, d ) A similar densitometric analysis to panel c, showing BDNF expression under slightly different experimental conditions. The molecular weight markers are indicated on the left, while the relative expression levels of BDNF are plotted in the treatment lanes. Figure 2 . The Brain-Derived Neurotrophic Factor (BDNF) before and after treatments . a ) Western blot of BDNF expression, with molecular weight markers (250–10 kDa) shown on the left. β-actin (~42 kDa) was used as a loading control for normalization, b ) Computerized analysis of Brain-Derived Neurotrophic Factor (BDNF) antibody protein expression level for samples, c ) Densitometric analysis of BDNF expression across experimental groups under different conditions, showing anti- Brain-Derived Neurotrophic Factor (BDNF) band calculation for samples, d ) A similar densitometric analysis to panel c, showing BDNF expression under slightly different experimental conditions. The molecular weight markers are indicated on the left, while the relative expression levels of BDNF are plotted in the treatment lanes. Western blotting findings BDNF, cleaved caspase-3

Under different treatment settings, the expression levels of the Brain-Derived Neurotrophic Factor (BDNF) antibody were analyzed (Figure 2). The control group exhibited a BDNF expression level of 46.01%, serving as the baseline for comparison. Exposure to radiation significantly decreased BDNF expression, with a lane percentage of 14.88%. This reduction highlights the detrimental effect of radiation on BDNF levels. After radiation exposure and MAG treatment, rats exhibited a BDNF expression level of 30.37%.

The expression levels of anti-cleaved caspase-3 were assessed in control and treated rat samples, with findings displayed as lane percentages (Figure 3). The control group had expression levels of 10.83% and 39.46%. The group exposed to radiation had significantly reduced expression levels of 9.37% and 15.73%. Finally, the cohort that had a particular therapy (MAG) exhibited expression levels of 10.69% and 27.69%. This indicates a potential protective role of antioxidants against radiation-induced cellular damage, a