Selenized Polysaccharides From Acanthopanax leucorrhizus: Synthesis, Characterization, and Cytotoxicity

ABSTRACT

Selenylation modification has been extensively developed as a method to enhance the biological effects of natural polysaccharides. In this study, a novel water‐soluble polysaccharide, designated as ALPS‐41, was isolated from the stem bark of A. leucorrhizus and subsequently selenized to form Se‐ALPS‐41, using the HNO₃–Na₂SeO₃ method. The physicochemical properties and structural features of both ALPS‐41 and Se‐ALPS‐41 were thoroughly characterized, using UV, Fourier transform infrared (FT‐IR), high‐performance gel‐permeation chromatography (HPGPC), HPLC, scanning electron microscopy (SEM), X‐ray diffraction (XRD), and NMR analyses. In vitro cytotoxicity was evaluated via Cell Counting Kit‐8 (CCK‐8) assays against human MCF‐7, HeLa, HepG2, and HEK293 cell lines. Chemical composition analysis revealed that the homogeneous ALPS‐41 had an average molecular weight of 437 kDa and was composed of glucose, galactose, and mannose in a molar ratio of 2.21:1.58:1.00. The selenium content and molecular weight of Se‐ALPS‐41 were determined to be 87.56 mg/g and 298 kDa, respectively. Cytotoxicity tests demonstrated that Se‐ALPS‐41 decreased the viability of MCF‐7 and HepG2 cells in a concentration‐dependent manner, while exhibiting no cytotoxic effect on HeLa and normal HEK293 cells. These findings suggest that selenium‐modified polysaccharides derived from A. leucorrhizus may serve as a potential selenium source with antitumor properties.

A novel water‐soluble polysaccharide ALPS‐41 was isolated from A. leucorrhizus stem bark and selenized to Se‐ALPS‐41. Characterized by multiple techniques, Se‐ALPS‐41 (Se content 87.56 mg/g, molecular weight 298 kDa) showed concentration‐dependent cytotoxicity against MCF‐7/HepG2 cells without harming HeLa/HEK293 cells, promising as potential antitumor selenium source.

1. Introduction

Over recent decades, natural polysaccharides, particularly those from medicinal plants, have attracted worldwide attention in biomedical research due to their diverse biological properties, low toxicity, biodegradability, and ability to interact with cellular pathways involved in apoptosis and immune responses [1, 2, 3]. Recent studies have shown that polysaccharides from various Acanthopanax species (Araliaceae family) exhibit a range of physiological and pharmacological effects, including free radical scavenging [4], antioxidant [5], anti‐aging [6], antiviral [7], immunomodulatory [8, 9], antitumor [10, 11], anti‐radiation [12], and blood glucose–lowering activities [13]. Given their confirmed bioactivities as well as low toxicity and side effects, Acanthopanax polysaccharides are considered promising candidates for clinical applications [14]. However, the bioactivity of natural polysaccharides is often limited by their structural constraints, prompting research into molecular modifications to enhance their efficacy. Among these, selenization—the incorporation of selenium into polysaccharide structures—has proven effective in enhancing bioactivity and broadening application prospects. This is attributed to two main factors: Selenium is an essential trace element involved in redox regulation and possesses inherent anticancer properties [15, 16], and selenium polysaccharides, as organic selenium compounds, exhibit dual and superior biological activity compared to either polysaccharides or selenium alone, with improved bioavailability [17]. Consequently, the selenization modification of polysaccharides has become a focus of research.

A. leucorrhizus is an endemic medicinal plant abundant in Gansu Province, China, known for its antibacterial, anti‐contracture, anti‐inflammatory, and antioxidant properties [18]. Previous phytochemical studies have mainly focused on its phenolic constituents and triterpenoids, whereas polysaccharides from A. leucorrhizus have received relatively little attention, especially regarding their antitumor activity [18]. Selenized polysaccharides from related species, such as A. senticosus, P. fortuneana, C. sinensis, C. sobolifera, M. sextelata, A. sphaerocephala, and T. caerulescens, have shown enhanced antitumor effects compared to their unmodified counterparts [16, 19, 20, 21, 22, 23], suggesting that similar modifications to A. leucorrhizus polysaccharides may yield innovative therapeutic agents.

In this study, a water‐soluble polysaccharide, designated ALPS‐41, was extracted and purified from the stem bark of A. leucorrhizus. Following this, it was then selenylated via the HNO₃–Na₂SeO₃ method to produce the selenized derivative Se‐ALPS‐41. The physicochemical properties and structural characteristics of both polysaccharides were determined, and their cytotoxic effects were evaluated in vitro. The aim of this study is to verify whether selenylation modification can enhance the cytotoxicity of ALPS‐41, broaden the understanding of A. leucorrhizus polysaccharides, and provide theoretical evidence for using selenylation to improve polysaccharide bioactivity.

2. Materials and Methods

2.1. Materials and Reagents

Stem bark of A. leucorrhizus was collected in September 2023 from Wushan County (Gansu Province, China) and authenticated by Prof. Xiaoqiang Guo of Longdong University. A voucher specimen (No. 20230922001) has been deposited at the Herbarium of the College of Agricultural and Biological Engineering, Longdong University, People's Republic of China. The collected stem bark was washed, shade‐dried, and ground into a 40‐mesh powder using a disintegrator.

MCF‐7 (human breast cancer), HeLa (human cervical cancer), HepG2 (human liver cancer), and HEK293 (human embryonic kidney 293) cell lines were obtained from PriCells Biomedical Technology Co. Ltd. (Wuhan, China). Cell Counting Kit‐8 (CCK‐8, BS350B, suitable for 500 tests, 5 × 1 mL) was purchased from Biosharp Life Science Co. Ltd. (Guangzhou, China). DEAE Cellulose DE‐52 (9013‐34‐7) and Sephadex G‐100 (9050‐94‐6, Mw 1000–100 000) were sourced from Yuanye Biotechnology Co. Ltd. (Shanghai, China). Sodium selenite (100 308), vitamin C (100 425), d‐glucose (130 809), d‐xylose (111 508), l‐rhamnose (111 683), d‐galactose (100 226), d‐mannose (140 651), d‐galacturonic acid (111 646), d‐glucuronic acid (140 648), trifluoroacetic acid (TFA, A501480), 5‐fluorouracil (140 857), 1‐phenyl‐3‐methyl‐5‐pyrazolone (PMP, 19735‐89‐8), acetonitrile (for HPLC, 130103), and dextran standards (Mw 5, 12, 25, 50, 80, 150, 280, 410, and 670 kDa, DXT 5‐670k) were all procured from Sinopharm Chemical Reagents Co. Ltd. (Shanghai, China). A selenium standard solution (1000 µg/mL, GSB 04‐1751‐2004) was provided by the National Non‐Ferrous Metals and Electronic Materials Analysis Testing Centers. Other analytical‐grade chemicals and solvents were purchased from Xi'an Chemical Co. (Xi'an, China).

2.2. Extraction and Purification of ALPS‐41

The stem bark of A. leucorrhizus was rinsed with running water and oven‐dried at 50 °C to constant weight. The dried bark was ground into a 40‐mesh powder using a disintegrator. The pretreated powder (300 g) was soaked three times in 95% ethanol (2.0 L each) for 24.0 h at room temperature to remove pigments and polyphenols. The air‐dried residue was then extracted three times with 2.0 L of ultrapure water at 80 °C for 5.0 h each. The combined extracts were concentrated to 25% of the original volume using a rotary evaporator at 60 °C, followed by centrifugation at 4000 rpm for 20 min. The supernatant was then collected and precipitated with a fivefold volume of anhydrous ethanol at 4 °C for 24 h. After centrifugation, the precipitate was redissolved in ultrapure water and deproteinated using the Sevag method [24]. Specifically, the polysaccharide solution was mixed with Sevag reagent (CHCl₃:n–C₄H₉OH = 4:1, V/V) in a 3:1 ratio (V/V), shaken vigorously for 30 min at room temperature and then centrifuged at 4000 rpm for 20 min. This deproteinization process was repeated three times. The supernatant was dialyzed against 100 volumes of ultrapure water for 72.0 h to further remove small molecular compounds (e.g., disaccharides or polyphenols). Finally, the dialyzate was lyophilized to obtain the crude polysaccharide ALPS (22.7 g).

ALPS was dissolved in ultrapure water and purified on a DEAE‐52 cellulose column (2.6 cm × 50 cm) equilibrated with ultrapure water. The sample was eluted stepwise with gradient NaCl solutions (0, 0.1, 0.2, and 0.3 M) at a flow rate of 1.0 mL/min. Fractions (5 mL/tube) were collected and monitored by absorbance at 490 nm, using the phenol–sulfuric acid method [25]. Four fractions (ALPS‐1, ALPS‐2, ALPS‐3, and ALPS‐4) were obtained. ALPS‐4 (eluted with 0.3 M NaCl) was further purified on a Sephadex G‐100 gel‐filtration column (1.6 cm × 50 cm) eluted with 0.1 M NaCl at 15.0 mL/h. The elution was monitored as above. The resultant fraction, named ALPS‐41, was collected, concentrated, dialyzed against ultrapure water, and lyophilized to yield the purified polysaccharide (98.8 mg).

2.3. Selenylation of ALPS‐41

Selenylation was performed using the HNO₃–Na₂SeO₃ method [25] with some modifications. Briefly, adhering to the previously optimized modification conditions, 0.5 g of ALPS‐41 was completely dissolved in 50.0 mL of 0.6% nitric acid with stirring. Then, 0.4 g of sodium selenite was added, and the reaction proceeded at 60 °C for 10.0 h under reflux. After cooling to room temperature, the pH was adjusted to 5–6 with a saturated sodium carbonate solution. The mixture was concentrated and dialyzed against ultrapure water using a 3000‐Da dialysis bag until no free sodium selenite was detected by the vitamin C method (indicated by the reaction solution turning from red to colorless) [26]. Finally, the polysaccharide solution was concentrated and freeze‐dried to obtain selenized ALPS‐41 (Se‐ALPS‐41).

2.4. Physicochemical Properties of ALPS‐41 and Se‐ALPS‐41

2.4.1. Determination of Carbohydrate and Selenium Content

Carbohydrate content was determined by the phenol–sulfuric acid method, using glucose as the standard [27]. Selenium content was quantified using an atomic fluorescence spectrometer (Model SA‐10, Beijing Jitian Instrument Co. Ltd.) [28]. A standard selenium solution was diluted with 5% HCl to concentrations of 0, 2.5, 5, 10, 20, and 40 µg/L. Fluorescence intensity corresponding to each concentration was measured, and a standard curve was plotted with selenium concentration (µg/L) on the X‐axis and fluorescence intensity on the Y‐axis. The regression equation was Y = 112.36X + 5.8965 (R ² = 0.9998).

Se‐ALPS‐41 (20 mg) was dissolved in 10.0 mL of ultrapure water. Then, 0.5 mL of this solution was mixed with 10.0 mL of HClO₄–HNO₃ (2:1, V/V) in a corked flask and digested at 4 °C for 12.0 h. The mixture was then heated to 180 °C, with mixed acid replenished as needed. Once the solution became clear, colorless, and emitted white smoke, it was concentrated to approximately 2.0 mL, cooled, mixed with 10.0 mL of 6 M HCl, reheated to 180 °C until the volume reduced to about 2.0 mL, cooled again, and diluted to 25.0 mL with 5% HCl. From this solution, 1.0 mL was diluted to 100.0 mL with 5% HCl as the sample solution. A blank solution was prepared similarly. Fluorescence intensities were measured, and selenium content (C, mg/g) was calculated using the regression equation and the following formula:

$$C=\frac{\left(X_1-X\right)\times V}{M\times 1000}$$ (1)

where X ₁ (µg/L) is the selenium concentration in the sample solution, X (µg/L) is the selenium concentration in the blank solution, V (mL) is the total volume, and M (mg) is the sample mass.

2.4.2. Analysis of Molecular Weights and Solubility

The homogeneity and molecular weight of ALPS‐41 were evaluated by high‐performance gel‐permeation chromatography (HPGPC) on an Agilent 1100 HPLC system equipped with a TSK‐GEL G‐4000PWXL column (7.8 mm × 30.0 cm) and an Agilent RID‐10A refractive index detector. The sample solution (2.0 mg/mL) was injected and eluted with 0.2 M NaCl at a flow rate of 0.6 mL/min. Data were processed using Agilent GPC software. Homogeneity was determined by the peak shape at 30 °C. Molecular weight was estimated using a standard calibration curve constructed with dextran standards (5, 12, 25, 50, 80, 150, 280, 410, and 670 kDa).

Solubility was assessed as described in the literature [29]. Briefly, 0.15 g of dried ALPS‐41 or Se‐ALPS‐41 was placed in centrifuge tubes, and 10.0 mL of ultrapure water was added. The mixture was agitated at room temperature for 30 min, with intermittent shaking every 5 min. After centrifugation at 8000 rpm for 20 min, the supernatant was removed. The centrifuge tubes were heated in an oven at 50 °C for 30 min, and solubility was determined through calculation.

2.4.3. Analysis of the UV Spectrum for ALPS‐41

ALPS‐41 was dissolved in distilled water to 1 mg/mL. The UV spectrum was scanned from 180 to 360 nm, using distilled water as the blank. Characteristic absorption peaks at 280 and 260 nm were used to ascertain the presence of proteins and nucleic acids [30].

2.4.4. Analysis of the Monosaccharide Composition in ALPS‐41

The monosaccharide composition of ALPS‐41 was determined by the PMP‐labeling method coupled with HPLC [31]. Briefly, 5.0 mg of ALPS‐41 was hydrolyzed with 3 mL of 2.0 M TFA at 120 °C for 3 h in a sealed glass tube. Excess TFA was removed by co‐evaporation with methanol under reduced pressure. The hydrolysate was PMP labeled by adding 0.6 M NaOH (2.0 mL) and 0.5 M PMP‐methanol solution (1.0 mL), followed by reaction at 70 °C for 2 h. The mixture was neutralized with 0.3 M HCl, centrifuged for 10 min, and the supernatant was filtered through a 0.45‐µm membrane for HPLC analysis. The solution (20 µL) was injected into an RP‐C₁₈ column (4.6 mm i.d. × 25 cm) and eluted with acetonitrile‐0.1 M phosphate buffer (20:80, pH = 6.8) at 1.0 mL/min. Standard monosaccharides (d‐glucose, d‐mannose, d‐galactose, l‐rhamnose, d‐xylose, l‐arabinose, d‐galacturonic acid, and d‐glucuronic acid) were similarly PMP‐labeled and analyzed. The monosaccharide composition of ALPS‐41 was identified by comparing retention times with standards.

2.5. Structure Characterization of ALPS‐41 and Se‐ALPS‐41

2.5.1. Scanning Electron Microscopy (SEM)

Dried ALPS‐41 or Se‐ALPS‐41 was mounted on a sample platform and coated. Surface morphology was examined using a field emission scanning electron microscope (GeminiSEM 300, Carl Zeiss, Germany) at 10.0 kV, 6.0 mm work distance, and 10.0 K magnification.

2.5.2. X‐Ray Diffraction (XRD) Analysis

The crystal structures of ALPS‐41 and Se‐ALPS‐41 were analyzed using an XRD. Data were collected over a 2θ range of 5°–90° at 40 kV, 30 mA, and a scan rate of 1°/min.

2.5.3. Thermogravimetric Analysis (TGA)

TGA was performed using a simultaneous thermal analyzer (TGA/DSC1/1100SF, Mettler Toledo, Switzerland). Dried samples (∼3.50 mg) were placed in an alumina crucible, with an empty crucible as reference. The heating rate was 10 °C/min from 25 °C to 800 °C under a nitrogen flow of 20 mL/min.

2.5.4. Stability Analysis

The mean diameter, zeta potential, and polydispersity index (PDI) of ALPS‐41 or Se‐ALPS‐41 were determined by dynamic light scattering (DLS) using a Zetasizer Nano ZS (NewBlue Technology Co. Ltd., Hangzhou, China). Sample stability was evaluated in deionized water at 1.0 mg/mL.

2.5.5. Fourier Transform Infrared (FT‐IR) Spectra Analysis

Functional groups of the polysaccharide were characterized using an FT–IR spectrophotometer (Model 8400S, Shimadzu, Japan). Briefly, after drying for 2.0 h in a drying oven, each sample (1.0 mg) was mixed with 100 mg of KBr, ground, and then pressed into a 1 mm pellet. Spectra were recorded from 400 to 4000 at 8 cm⁻¹ resolution with 32 scans. Data were analyzed using Nicolet Omnic 8.0 software.

2.5.6. NMR Spectra Analysis

Dried Se‐ALPS‐41 (30 mg) underwent deuterium exchange in D₂O (99.9%) three times, followed by lyophilization. The sample was dissolved in 0.5 mL of D₂O, heated to 85 °C for 3.0 h, and analyzed by ¹³C‐NMR (AMX‐500, Bruker, Switzerland), using TMS as the internal standard and D₂O as the solvent at 25 °C.

2.6. Cytotoxicity Evaluation of ALPS‐41 and Se‐ALPS‐41

2.6.1. Cell Cultures

MCF‐7, HeLa, HepG2, and HEK293 cells were incubated in DMEM complete medium supplemented with 10% FBS and 1% penicillin‐streptomycin at 37 °C in a 5% CO₂ humidified incubator. Upon reaching 75% confluence, adherent cells were gently scraped, collected, and centrifuged at 800 rpm for 5 min for subsequent experiments.

2.6.2. CCK‐8 Assay

Cytotoxicity was evaluated using the CCK‐8 assay [32]. Briefly, 100 µL of cell suspension was seeded into 96‐well plates and treated with various concentrations of ALPS‐41 or Se‐ALPS‐41 for 24 h. Then, 10 µL of CCK‐8 reagent was added to each well and incubated for 2 h. Absorbance at 460 nm was measured using a microplate reader (BioTek, Epoch, USA). Sterile ultrapure water served as the negative control, DMEM medium without polysaccharide as the blank, and 5‐fluorouracil (5‐Fu) as the positive control. Cell viability was calculated as

$$\text{Cell viability}=\frac{A_2-A_0}{A_1-A_0}\times 100\%$$ (2)

where A ₀, A ₁, and A ₂ are the absorbance of the negative control, blank, and test groups, respectively.

2.7. Statistical Analysis

All tests were performed in triplicate, and data are presented as mean ± SD. Statistical analyses were conducted using Origin 2018, and significance (p < 0.05) was assessed by ANOVA with SPSS 22.0.

3. Results and Discussion

3.1. Isolation and Purification of ALPS‐41

The essential processes of extraction, purification, and selenylation of ALPS‐41 are schematically illustrated in Figure 1.

FIGURE 1.

Extraction, purification, and selenylation processes of ALPS‐41.

Crude ALPS was extracted from A. leucorrhizus stem bark and fractionated on a DEAE‐52 column using stepwise NaCl solutions (0, 0.1, 0.2, and 0.3 M), yielding four fractions (ALPS‐1, ALPS‐2, ALPS‐3, and ALPS‐4, Figure 2A). ALPS‐4 was further purified on a Sephadex G‐100 column with deionized water, yielding a singular elution peak (Figure 2B), designated ALPS‐41 (98.8 mg). The UV spectrum of ALPS‐41 showed a maximum absorption near 200 nm (Figure 2C), the characteristic of polysaccharides, with no absorbance at 260 or 280 nm, indicating the absence of protein or nucleic acid impurities [30, 33]. Thus, the purification methods were effective. The HPGPC profile of ALPS‐41 displayed a single, symmetrical, narrow peak (Figure 2D), confirming that ALPS‐41 is a highly pure, homogeneous polysaccharide suitable for selenylation.

FIGURE 2.

Isolation and purification of ALPS‐41: (A) stepwise elution curve of ALPS on DEAE‐52 column; (B) elution profile of ALPS‐41 on Sephadex G‐100 column; (C) UV spectra of ALPS‐41; and (D) HPGPC profile of ALPS‐41.

3.2. Physicochemical Properties of ALPS‐21 and Se‐ALPS‐21

3.2.1. Solubility, Particle Size, and Zeta Potential

Key physicochemical properties of ALPS‐41 and Se‐ALPS‐41 are listed in Table 1. Se‐ALPS‐41 showed a solubility of 90.45% ± 0.98%, higher than that of ALPS‐41 (78.85% ± 1.16%). Polysaccharide solubility is influenced by molecular weight, with high molecular weights reducing solubility due to increased intermolecular interactions [34]. Thus, the lower molecular weight of Se‐ALPS‐41 contributes to its higher solubility. Selenium polysaccharides are polar macromolecules with good water solubility [35]. The particle size of polysaccharides directly affects their physical stability; smaller particles improve dispersion stability. As depicted in Table 1, selenization reduced the particle size from 198.12 ± 2.32 nm (ALPS‐41) to 143.31 ± 1.31 nm (Se‐ALPS‐41), thereby enhancing dispersion stability in aqueous solution. The zeta potential also influences the stability of particles within the dispersion through interparticle electrostatic repulsion [36]. After selenization, the zeta potential changed from −0.19 ± 0.02 mV (ALPS‐41) to −15.41 ± 0.29 mV (Se‐ALPS‐41). The higher absolute value of the zeta potential indicates better dispersion and stability for Se‐ALPS‐41. In summary, selenization significantly improves the stability of polysaccharide.

TABLE 1.

Main physicochemical parameters of ALPS‐41 and Se‐ALPS‐41.

Sample	Carbohydrate content (%)	Selenium content (mg/g)	Molecular weight (kDa)	Solubility (%)	Particle size (nm)	Zeta (mV)
ALPS‐41	96.81 ± 0.36	—	437	78.85 ± 1.16	198.12 ± 2.32	−0.19 ± 0.02
Se‐ALPS‐41	71.45 ± 0.31	87.56 ± 0.45	298	90.45 ± 0.98	143.31 ± 1.31	−15.41 ± 0.29

3.2.2. Carbohydrate and Selenium Contents

As shown in Table 1, the carbohydrate contents of ALPS‐41 and Se‐ALPS‐41 were 96.81% ± 0.36% and 71.45% ± 0.31%, respectively, and the selenium content of Se‐ALPS‐41 was 87.56 ± 0.45 mg/g.

3.3. Structural Characterization of ALPS‐41 and Se‐ALPS‐41

3.3.1. SEM Analysis

SEM images of ALPS‐41 and Se‐ALPS‐41 are shown in Figure 3A,B, respectively. The surface of ALPS‐41 is covered with minute bumps, whereas Se‐ALPS‐41 exhibits a sheet‐like or fragmented accumulation with a smooth texture. This irregular surface distribution in Se‐ALPS‐41 may result from intermolecular force (mainly van der Waals force) between selenium and polysaccharides.

FIGURE 3.

Structural characterization of ALPS‐41 and Se‐ALPS‐41: (A) SEM image of ALPS‐41; (B) SEM image of Se‐ALPS‐41; (C) TGA curves of ALPS‐41 and Se‐ALPS‐41; (D) XRD curves of ALPS‐41 and Se‐ALPS‐41; (E) HPLC chromatograms of standard monosaccharides and component monosaccharides of ALPS‐41; (F) FT‐IR spectra of ALPS‐41 and Se‐ALPS‐41; and (G) ¹³C‐NMR spectra of Se‐ALPS‐41.

3.3.2. Molecular Weight

HPGPC analysis showed a significant (p < 0.05) 31.8% reduction in molecular weight from 437 kDa (ALPS‐41) to 298 kDa (Se‐ALPS‐41) (Table 1). This decrease suggests structural modifications, possibly due to acid‐catalyzed hydrolysis of glycosidic bonds during selenization [37, 38]. The notable disparity in molecular weight is particularly significant, given that polysaccharides with higher molecular weight (>50 kDa) have been well documented in pharmacological research to enhance antitumor efficacy [39].

3.3.3. Thermogravimetric Analysis

Thermal stability of polysaccharides was assessed by TGA. The TGA curves (Figure 3C) showed initial mass losses (25–215 °C) of 12.52% for Se‐ALPS‐41 and 15.81% for ALPS‐41, attributed to the evaporation of self‐bound water. Further decomposition (215–420 °C) resulted in mass losses of 63.75% for Se‐ALPS‐41 and 77.46% for ALPS‐41, corresponding to the degradation of polysaccharide chains [40]. Pyrolysis of the polysaccharide skeleton in this range led to mass losses of approximately 61.65% for ALPS‐41 and 51.23% for Se‐ALPS‐41. The lower mass loss for Se‐ALPS‐41 may be due to selenite group incorporation enhancing thermal resistance. From 420 °C to 800 °C, both polysaccharides maintained stability, likely due to the carbonization of the polysaccharide. In summary, Se‐ALPS‐41 exhibited better thermal stability than ALPS‐41.

3.3.4. XRD Analysis

Figure 3D displays the dispersed absorption peaks at 21.68 (ALPS‐41) and 20.24 (Se‐ALPS‐41). ALPS‐41 had a weak diffraction peak at 29.42°, absent in Se‐ALPS‐41, suggesting crystallinity changes and possible formation of a new compound after selenization. Additionally, the weak diffraction peaks indicate that neither ALPS‐41 nor Se‐ALPS‐41 forms large single crystals [41], likely retaining an amorphous structure.

3.3.5. Monosaccharide Composition of ALPS‐41

The monosaccharide composition of ALPS‐41 was determined by PMP‐HPLC method. On the basis of the retention time and peak area of each standard (Figure 3E), ALPS‐41 was identified as a neutral heteropolysaccharide composed of glucose (Glc), galactose (Gal), and mannose (Man) in a molar ratio of 2.21:1.58:1.00.

3.3.6. FT‐IR Analysis

FT‐IR spectra of ALPS‐41 and Se‐ALPS‐41 were recorded in the range of 400–4000 cm⁻¹, as depicted in Figure 3F. The spectra exhibited the characteristic absorptions of polysaccharides, with intense broad peaks at around 3441 cm⁻¹ and weaker peaks at about 2921 cm⁻¹, which ascribed to the stretching vibrations of O─H and aliphatic C─H, respectively. The peaks at 1416 cm⁻¹ were attributed to bending vibrations of C─H. Additionally, the peaks at 1641 cm⁻¹ indicated the presence of bound water [42]. Specifically, the faint peak at nearly 1108 cm⁻¹ and the strong peak at around 1028 cm⁻¹ suggested the stretching vibrations of the side group C─O─H and the glycosidic bond C─O─C, indicating the presence of a pyranose ring [43]. Notably, the absorption peaks at 768 and 887 cm⁻¹ belong to α‐ and β‐type glycosidic bonds, respectively, indicating the presence of both α‐ and β‐type glycosidic bond‐linked pyranose rings [44]. Crucially, the spectrum of Se‐ALPS‐41 exhibited a sharp peak at around 640 cm⁻¹, corresponding to Se─O─C stretching vibrations [45], and a well‐defined peak at 1074 cm⁻¹ attributed to Se = O asymmetrical stretching vibrations [46], which were absent in ALPS‐41. These results indicated that some hydroxyl groups on ALPS‐41 were successfully substituted by selenyl groups during selenylation [47].

3.3.7. ¹³C‐NMR Analysis

The ¹³C‐NMR spectra of ALPS‐41 and Se‐ALPS‐41 are presented in Figure 3G. For ALPS‐41, the signals were clearly observed in the range of δ _C 60–110 ppm, consistent with the characteristic chemical shifts of polysaccharides [44]. Among these signals, the peaks at 95.8, 97.5, 98.3, and 98.8 ppm were typically attributed to four anomeric carbons of α‐configuration, whereas those at 101.6, 102.3, and 102.6 ppm corresponded to the three anomeric carbons of β‐configuration [48]. These results indicate that ALPS‐41 contains both α‐ and β‐configurations, which aligns with the FT‐IR analysis. The signals ranging from 62.2 to 81.6 ppm were attributed to C2–C6 of the sugar residues. In Se‐ALPS‐41, the peak observed at 62.8 ppm was assigned to the O‐6‐substituted carbons, indicating reaction at the O‐6 position [49]. From the analysis of ¹³C‐NMR, the signal intensity of O‐substituted carbons demonstrated that C‐6 substitution was predominant in Se‐ALPS‐41 [50]. This dominance can be attributed to the relatively high reactivity of the hydroxyl group at the C‐6 position of the polysaccharide. Under nitric acid catalysis, sodium selenite reacts with the hydroxyl group at the C‐6 position to form selenium‐containing polysaccharide [51]. In summary, the ¹³C‐NMR spectra confirmed the successful selenylation of ALPS‐41 and provided information about the substitution locations.

3.4. Cytotoxicity Assessment

The cytotoxic effects of ALPS‐41 and Se‐ALPS‐41 on three cancer cell lines (MCF‐7, HeLa, and HepG2) and a normal cell line (HEK293) were evaluated by CCK‐8 assay. As depicted in Figure 4, 5‐Fu (positive control) exhibited dose‐dependent inhibitory effects on all tested cell lines, with the inhibition rates of 44.32% ± 1.42%, 48.02% ± 1.53%, 56.49% ± 1.98%, and 66.11% ± 2.36% at a concentration of 1.0 mg/mL, respectively. Likewise, ALPS‐41 demonstrated only weak cytotoxicity against MCF‐7 cells, with an inhibition rate of 9.37% ± 0.34% at 1.0 mg/mL. In contrast, Se‐ALPS‐41 exhibited significant cytotoxicity against MCF‐7 (39.37% ± 1.04%) and HepG2 (32.06% ± 0.95%) cells in a dose‐dependent manner at 1.0 mg/mL but showed no cytotoxicity against HeLa or HEK293 cells at the tested concentrations. These results indicate that Se‐ALPS‐41 can significantly reduce the viability of MCF‐7 and HepG2 cells, while remaining safe for normal HEK293 cells. This study provides evidence for the first time that polysaccharides from A. leucorrhizus exhibit cytotoxic activity, and that selenium modification can significantly enhance the cytotoxic potency of Acanthopanax polysaccharide.

FIGURE 4.

Cytotoxicity of ALPS‐41 and Se‐ALPS‐41: (A) effect on MCF‐7 growth rate; (B) effect on HeLa growth rate; (C) effect on HepG2 growth rate; and (D) effect on HEK293 growth rate.

4. Conclusion

ALPS‐41 is a neutral polysaccharide isolated and purified from A. leucorrhizus, consisting of glucose, galactose, and mannose with a molar ratio of 2.21:1.58:1.00. It was subjected to selenylation modification via the HNO₃─Na₂SeO₃ method to obtain Se‐ALPS‐41. FT‐IR analysis of Se‐ALPS‐41 revealed two novel absorption peaks at approximately 640 and 1074 cm⁻¹, confirming the successful substitution of hydroxyl groups with selenyl groups. Furthermore, ¹³C‐NMR signals verified that O‐substitution predominantly occurred at the C‐6 position of ALPS‐41. Compared with ALPS‐41, Se‐ALPS‐41 exhibited a significantly higher selenium content (87.56 ± 0.45 mg/g), along with reduced molecular weight, smaller particle size, improved water solubility, and enhanced thermal stability. In vitro assays demonstrated that Se‐ALPS‐41 effectively inhibited the proliferation of human MCF‐7 and HepG2 cells in a dose‐dependent manner, displaying remarkably stronger cytotoxic activity than ALPS‐41, while remaining non‐toxic to HeLa and normal HEK293 cells. These results demonstrate that selenylation modification can substantially enhance the solubility, dispersion stability in aqueous solutions, thermal stability, and antitumor cytotoxicity of ALPS‐41. These findings not only provide a promising strategy for enhancing the bioactivity of polysaccharides from A. leucorrhizus, but also highlight Se‐ALPS‐41 as a potential candidate for the development of selenium‐based nutritional supplements and antitumor agents.

However, the diversity of known selenium‐containing polysaccharides—both domestic and international—remains limited. Additionally, their precise chemical structures and in vivo mechanisms of action have not been fully elucidated, particularly for natural selenium polysaccharide. Consequently, further in‐depth investigation in these areas is imperative.

Author Contributions

Haobin Hu: conceptualization, supervision, funding acquisition, project administration. Zhenyu Cheng: resources, data analysis. Haipeng Liang: validation, methodology. Minghu Han: visualization, data curation. Yanan Hu: investigation, writing – original draft. Yun Wu: formal analysis, software. Yufeng Wang: statistical analysis, writing – review and editing. All authors contributed to the article and approved the submitted version.

Funding

This work was financially supported by the National Natural Science Foundation of China (Grant 22267013) and the Horizontal Scientific Research Project of Longdong University (Grant HXZK2275).

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

No data were used for the research described in the article.