A structure-maintaining type side chain seleniumized rhodiola fruit pectin polysaccharide, and a preparation method and application thereof

Abstract

This invention discloses a structure-preserving side-chain selenized Rhodiola rosea pectin polysaccharide, its preparation method, and its applications. The method uses a eutectic solvent (DES) as a homogeneous reaction medium to fully dissolve and extend the chain segments of the Rhodiola rosea pectin polysaccharide, which possesses multiple structural domains (HG-RG-I / AG), high branching, and high molecular weight characteristics, under mild acidic conditions. This allows for sufficient contact and reaction with selenite, introducing selenium-containing groups while inhibiting excessive glycosidic bond breakage and maintaining a high weight-average molecular weight. By selecting a slightly acidic reaction condition with an apparent pH of 4.5, this invention achieves a good balance between selenium content and reaction mildness. The resulting selenized Rhodiola rosea polysaccharide has a selenium content of 19.4 ± 3.1 mg / g, a weight-average molecular weight retention rate of 93.8%, and a saturated solubility 116.9% higher than the original sugar. It exhibits significantly enhanced in vitro chemical antioxidant capacity and cellular-level antioxidant stress protection.

Description

Technical Field

[0001] This invention relates to the field of natural polysaccharide chemical modification technology, and more specifically to a structure-preserving side-chain selenized Rhodiola rosea pectin polysaccharide, its preparation method, and its application. Background Technology

[0002] Rhodiola rosea polysaccharides are one of the important material bases for Rhodiola rosea's immune-regulating, antioxidant, and anti-fatigue biological effects. Monosaccharide composition and NMR analysis show that Rhodiola rosea polysaccharides belong to acidic pectin-type heteropolysaccharides with HG (homocalacturonic acid polysaccharide), RG-I (rhamnogalacturonic acid polysaccharide I), and AG (arabinogalactan) side chain structures. More specifically, its structural characteristics are not simply the coexistence of HG, RG-I, and AG domains, but rather a structural combination with an HG-RG-I complex acidic backbone as the main body, Gal / Ara-enriched AG-type side chains as the main branched structure, coupled with high molecular weight characteristics. Among them, the HG region constitutes an important acidic backbone of pectin-type polysaccharides, while the RG-I region and its connected neutral sugar side chains such as AG enable the polysaccharide to form a highly branched complex spatial structure. The different structural domains are not isolated from each other, but jointly determine the molecular weight, chain conformation, aggregation state, aqueous dispersion behavior, and potential biological recognition ability of the polysaccharide.

[0003] Under the synergistic effect of the aforementioned high molecular weight backbone and highly branched side chains, the HG region and the RG-I / AG enriched region may form a relatively dense intramolecular or intermolecular associated structure through segment entanglement, hydrogen bonding, ionic interactions, and other non-covalent interactions. On the one hand, this complex structure formed by the synergistic effect of multiple structural domains may be an important structural basis for the biological activity of Rhodiola rosea polysaccharides; on the other hand, excessive entanglement and aggregation will reduce the contact between the polysaccharide and water molecules, resulting in slow dissolution rate, poor high-concentration dispersion stability, and limiting the exposure of some potential active structures. This may also reduce the accessibility of reaction reagents to polysaccharide segments and reactive sites. Therefore, the need for modification of Rhodiola rosea polysaccharides is not simply due to the presence of HG, RG-I, or AG structures, but mainly related to the dense aggregated state formed by its multiple structural domains, high branching, and high molecular weight.

[0004] The weight-average molecular weight of the Rhodiola rosea polysaccharide used in this study was approximately 1.40 × 10⁻⁶. 6Da. This high molecular weight characteristic is a comprehensive manifestation of its complex main chain, branched side chains, and multi-level aggregated structure. Such structures are quite sensitive to chemical reactions: excessively strong acidity or reaction conditions may cause glycosidic bond breakage, a decrease in the molecular weight of the main chain, or changes in the side chain structure; however, overly mild reaction conditions may lead to insufficient introduction of selenium-containing groups. Therefore, simply pursuing an increase in the degree of reaction or a decrease in molecular weight may destroy the original structural and functional basis of Rhodiola rosea polysaccharides.

[0005] Existing polysaccharide selenization methods typically require a certain acidic environment to promote the reaction. When the selenization reaction occurs simultaneously with glycosidic acid-catalyzed hydrolysis, the problem of increased selenium content but a significant decrease in polysaccharide molecular weight is prone to occur. For Rhodiola rosea polysaccharides with multiple HG-RG-I / AG domains and high molecular weight characteristics, it is necessary to select a reaction system that matches its structural tolerance: ensuring sufficient contact of reactants within the same system while avoiding excessive damage to the main chain and side chains due to excessive acidity.

[0006] Homogeneous reaction systems can promote the dissolution and chain extension of high molecular weight Rhodiola rosea polysaccharides, allowing for sufficient contact between the polysaccharides and selenium-containing reactants. However, the homogeneous state itself cannot guarantee the preservation of the polysaccharide structure, and the acidity of the reaction may still affect the stability of glycosidic bonds. Therefore, the key problem to be solved in this field is: how to screen suitable homogeneous reaction conditions and slightly acidic reaction ranges based on the unique uronic acid content, monosaccharide composition, multi-domain structure, and high molecular weight characteristics of Rhodiola rosea pectin-type polysaccharides, so as to reduce overall structural damage while introducing selenium-containing groups, and moderately loosen the entanglement and aggregation between the HG region and the RG-I / AG enriched region, thereby improving its water solubility, dispersibility, and antioxidant function. Summary of the Invention

[0007] In view of this, the present invention targets Rhodiola rosea pectin-type polysaccharides with multiple HG-RG-I / AG domains, high branching, and high molecular weight. The structural features of interest in this invention are not merely the simultaneous presence of HG, RG-I, and AG domains, but rather the polysaccharide's structure, characterized by a predominantly HG-RG-I complex acidic backbone, with Gal / Ara-enriched AG-type side chains as the main branching structure, and strong segmental entanglement and aggregation formed synergistically by the high molecular weight backbone and the enriched side chains. This structural combination, on the one hand, reduces the accessibility of water molecules and selenium-containing reactants to the polysaccharide segments, and on the other hand, makes the HG backbone and RG-I / AG-enriched regions more susceptible to structural damage under acidic reaction conditions. This paper addresses the challenge of selecting homogeneous selenization conditions that match the structure's tolerance, ensuring sufficient contact between the polysaccharide and selenium-containing reactants, maintaining a high level of selenium introduction while inhibiting excessive glycosidic bond breakage, reducing the risk of damage to the HG backbone and RG-I / AG enrichment region, and promoting the unwinding of excessive entanglement and aggregation between different structural domains. It presents a structure-preserving side-chain selenized Rhodiola rosea pectin polysaccharide, its preparation method, and its applications.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] A method for preparing a structure-preserving side-chain selenized Rhodiola rosea pectin polysaccharide includes the following steps: S1. Constructing a eutectic solvent system: Mix hydrogen bond acceptors and hydrogen bond donors at a preset molar ratio, heat and stir until clear and transparent to obtain a eutectic solvent system; the apparent pH of the eutectic solvent system is controlled between 3.5 and 5.5; S2. Homogeneous dissolution: Rhodiola polysaccharide is added to the eutectic solvent system preheated to 60°C and stirred for 2-4 hours under nitrogen protection until a transparent homogeneous solution is formed. S3, Selenization reaction: Add selenite to the transparent homogeneous solution obtained in step S2, and react at 55-65℃ for 3-5 hours to obtain the selenization reaction solution. S4. Purification: Cool the selenization reaction solution obtained in step S3 in an ice bath, add a precipitant to precipitate, collect the precipitate by centrifugation, and then purify it by dialysis after redissolving in deionized water to obtain selenized Rhodiola rosea polysaccharide.

[0010] Preferably, in step S1, the eutectic solvent system is selected from the choline chloride / ethylene glycol system or the choline chloride / glycerol system.

[0011] Preferably, in the choline chloride / ethylene glycol system or the choline chloride / glycerol system, the molar ratio of choline chloride to hydrogen bond donor is 1:2, and the apparent pH is adjusted to 3.5, 4.5 or 5.5 by adding imidazole or triethylamine.

[0012] Preferably, in step S1, the apparent pH of the eutectic solvent system is 4.5.

[0013] Preferably, in step S4, the precipitant is anhydrous ethanol or acetone, and the added volume is 3 times the volume of the reaction liquid; the dialysis is performed using a dialysis bag with a molecular weight cutoff of 3500 Da, and dialysis is performed with running water for 48 hours.

[0014] As can be seen from the above technical solution, compared with the prior art, the present invention has the following technical effects: 1. This invention achieves selenium-containing modification while reducing polysaccharide backbone degradation and maintaining a high weight-average molecular weight through synergistic regulation of a homogeneous reaction system and experimentally screened slightly acidic conditions. At the same time, it regulates the aggregation state and aqueous dispersion behavior, thereby improving solubility and enhancing in vitro chemical antioxidant capacity and cellular antioxidant stress resistance.

[0015] 2. This invention enhances the interaction between Rhodiola rosea polysaccharide and its target sites by introducing selenium-containing groups to regulate the local conformation and action sites of the polysaccharide side chains. Therefore, the enhanced activity stems not only from improved solubility and dispersibility but also from the structural functionalization imparted by selenization modification. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0017] Figure 1 Elution curves of Rhodiola polysaccharide (RRP) on Sephadex G-100 dextran gel column chromatography; Figure 2 This is the standard curve for galacturonic acid. Figure 3 HPLC chromatogram for PMP derivatization determination of Rhodiola rosea polysaccharide (RRP) monosaccharide composition; Figure 4 Here is the NMR spectrum of the polysaccharide, where (A) is... 1 The H NMR spectrum, (B) is... 13 C10 NMR spectrum, (C) is 1 H- 1 The H COSY spectrum, (D) is... 1 H- 13 C HSQC spectrum, (E) is 1 H- 13 C HMBC spectrum; Figure 5 SEM image of Rhodiola rosea polysaccharide (RRP); Figure 6 2D and 3D height maps of Rhodiola polysaccharide (RRP) AFM; Figure 7 The results of selenium content measurement in products of different reaction systems; Figure 8 Results of selenium substitution degree detection in products of different reaction systems; Figure 9 Results of uronic acid content detection at different pH values; Figure 10 For comparison of the infrared spectra of selenized polysaccharide Se-RRP and RRP; Figure 11 Comparison of UV spectra of selenized polysaccharide Se-RRP and RRP; Figure 12 Comparison of the solubility states of selenized polysaccharide Se-RRP and RRP at different concentrations; Figure 13 SEM image of Rhodiola rosea polysaccharide (Se-RRP) after selenization modification; Figure 14 AFM 2D and 3D height maps of selenized Rhodiola rosea polysaccharide (Se-RRP); Figure 15 The results of DLS detection of RRP and Se-RRP aqueous solutions; Figure 16 The in vitro free radical scavenging capabilities of Rhodiola rosea polysaccharide (RRP) and selenized polysaccharide (Se-RRP) are shown, where (A) represents the ABTS free radical scavenging rate and (B) represents the DPPH free radical scavenging rate. Figure 17 The effects of different concentrations of Rhodiola rosea polysaccharide (RRP) and selenized polysaccharide (Se-RRP) on the activity of H9c2 rat cardiomyocytes; Figure 18 To detect the level of reactive oxygen species (ROS) and their fluorescence intensity distribution in cells of each treatment group by flow cytometry; Figure 19 To detect the level of reduced glutathione (GSH) and its fluorescence intensity distribution in cells of each treatment group by flow cytometry; Figure 20 The molecular dynamics simulation analysis of the binding of RRP and Se-RRP ligands to Keap1 protein at 100 ns is shown in the figures. (A) is the RMSD curve of the protein backbone, (B) is the RMSF flexibility of the Keap1 Kelch domain residues, (C) is the radius of gyration of the complex (Rg), (D) is the solvent-accessible surface area (SASA), (E) is the real-time change of the number of hydrogen bonds at the ligand-Keap1 interface, and (F) is the comparison of MM / PBSA binding free energy. Detailed Implementation

[0018] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] This invention discloses a method for preparing a structure-preserving side-chain selenized Rhodiola rosea pectin polysaccharide.

[0020] First, the original structure of Rhodiola rosea polysaccharide was analyzed to provide a foundation for subsequent analysis: Polysaccharide homogeneity Rhodiola rosea polysaccharide (RRP) was purified and its homogeneity was identified using Sephadex G-100 column chromatography. The phenol-sulfuric acid method (490 nm) showed that the elution curve exhibited only a single, symmetrical sugar absorption peak (near tube 11), without any extraneous peaks or shoulder peaks. This elution behavior indicates that the purified RRP has good molecular weight distribution uniformity and is a homogeneous polysaccharide with a single component.

[0021] Glucuronic acid content The uronic acid content in polysaccharides was determined using the m-hydroxybiphenyl method. Accurately weigh the polysaccharide sample and dissolve it in pure water to prepare the test solution. Transfer 0.5 mL of the sample solution or a series of galacturonic acid (GalA) standard solutions to colorimetric tubes. Add 3 mL of borax-concentrated sulfuric acid solution (0.478 g / 100 mL) under ice bath conditions, mix well, and heat in a boiling water bath for 5 min, followed by rapid cooling in an ice bath. Add 50 μL of m-hydroxybiphenyl reagent, mix thoroughly, and react at room temperature in the dark for 20 min. Measure the absorbance at 520 nm. Plot a standard curve using GalA and calculate the uronic acid content in the sample.

[0022] Plotting the mass concentration of galacturonic acid (GalA) on the x-axis and the absorbance value on the y-axis, the standard curve equation is obtained as: y = 0.0085x - 0.0071, R0 2 = 0.995. This indicates a good linear relationship within the measurement range. The absorbance value of the sample solution was 0.1010. Substituting this into the regression equation, the calculated galacturonic acid concentration was 7.14 μg / mL. The actual galacturonic acid content in the sample was then calculated to be 22.80% (see [reference]). Figure 2 ).

[0023] Monosaccharide composition Monosaccharide composition analysis revealed that the polysaccharide is mainly composed of galactose (Gal), galacturonic acid (GalA), arabinose (Ara), and rhamnose (Rha), with small amounts of mannose (Man), glucose (Glc), xylose (Xyl), and fucose (Fuc). The high content of GalA and Rha confirms that this polysaccharide possesses a typical pectin structure, with the main chain backbone composed of the HG homogeneous domain and the RG-I heterogeneous domain. Furthermore, the high molar ratio of Gal and Ara further indicates that abundant arabinogalactan (AG) or arabinogalactan / polygalactose side chains are grafted onto the Rha residues of the RG-I backbone. These monosaccharide composition characteristics clearly confirm that this polysaccharide is a typical branched RG-I type pectin heteropolysaccharide (see [link to article]). Figure 3 ).

[0024] NMR spectroscopy analysis Based on the monosaccharide composition and one-dimensional and two-dimensional NMR spectra, this polysaccharide should be classified as a branched pectin heteropolysaccharide rich in RG-I domains. 1 In 1H NMR, multiple anomeric hydrogen signals at δ 5.35, 5.17, 4.79, 4.72, and 4.43 ppm indicate the presence of various sugar residues and different glycosidic configurations in the sample. The strong signal near δ 5.3 ppm is generally associated with α-configuration sugar residues and can be attributed to residues such as α-GalA, α-Rha, or α-Ara; while the signal in the δ 4.4–4.8 ppm region suggests the presence of β-configuration Gal or other side-chain sugar residues. In 13C NMR, a significant carboxyl carbon signal appears in the δ 170–181 ppm region. Combined with the high GalA content in the monosaccharide composition, this indicates that the polysaccharide contains abundant galacturonic acid, which is important evidence for pectin-like polysaccharides. Multiple anomeric carbon signals in the δ 92–105 ppm region further confirm its complex sugar residue composition, consistent with the structural characteristics of RG-I pectin heteropolysaccharides.

[0025] Two-dimensional spectra further support the above judgment. Multiple typical anodic correlation peaks were observed in the HSQC, such as δH / δC 5.34 / 92.06, 5.34 / 99.51, 4.92 / 98.00, 4.58 / 104.26, and 4.57 / 95.82, indicating that the polysaccharide contains at least different residues such as α-GalA, α-Rha, α-Ara, and β-Gal. Correlation peaks such as 5.24 / 3.25, 4.94 / 3.54, 4.58 / 3.18, 4.22 / 4.22, and 3.58 / 5.34 in the COSY spectrum reflect the coupling relationships between H-1 / H-2, H-2 / H-3, or adjacent protons within the sugar ring, indicating that these anodic signals indeed originate from the polysaccharide backbone and side-chain sugar residues. Long-range correlation peaks in HMBC, such as δH / δC 5.33 / 103.55, 5.47 / 92.06, 3.80 / 103.56, and 3.59 / 105.18, indicate the presence of glycosidic linkages between sugar residues. These peaks could support the structural features of alternating GalA and Rha forming the RG-I backbone, as well as Ara / Gal side links branching onto Rha residues.

[0026] Furthermore, the methyl carbon signal near δ 16–18 ppm and the low-field methyl correlation signal in 1H correspond to the C-6 methyl group of Rha, providing important evidence for the presence of rhamnose residues in the RG-I structure. Based on the monosaccharide composition and NMR results, this polysaccharide should possess a pectin backbone composed of a homogeneous HG galacturonic acid region and a heterogeneous RG-I region, with the RG-I region rich in Rha and numerous Gal / Ara arabinogalactan or arabinogalactan side chains grafted at the Rha sites. Therefore, this polysaccharide can be preliminarily identified as a highly branched, acidic pectin-type heteropolysaccharide dominated by the RG-I domain (see [link to NMR analysis]). Figure 4 ).

[0027] SEM and AFM morphology analysis Scanning electron microscopy (SEM) images show that natural Rhodiola rosea polysaccharide (RRP) exhibits a continuous, dense, and thick sheet-like and blocky structure. At different magnifications, the sample surface appears relatively smooth overall, with clear edges and no obvious pores. This morphological characteristic indicates the presence of strong hydrogen bonding interactions and chain segment entanglement between and within the native polysaccharide molecules, promoting the disordered stacking of polysaccharide chains and the formation of a highly compact aggregated microstructure (see [link to SEM image]). Figure 5 ).

[0028] Atomic force microscopy (AFM) 2D and 3D height images revealed that natural Rhodiola rosea polysaccharide (RRP) forms high-density, dome-shaped or spherical aggregates on the mica surface. Data showed that the aggregate height reached 20–60 nm (up to approximately 63.4 nm), significantly exceeding the theoretical diameter of a single polysaccharide chain (approximately 0.1–1.0 nm). This further confirms the existence of strong hydrogen bonds between and within the native polysaccharide molecules, leading to their high entanglement in aqueous solution and a tendency to self-assemble into large spherical molecular aggregates (see [link to AFM image]). Figure 6 ).

[0029] Selenization reaction process under different conditions: This reaction first constructs three DES systems with different pH values ​​and hydrogen bonding strengths: a strongly acidic system using ChCl / malic acid in a 1:1 molar ratio, with pH controlled at 2.0–3.0; a mildly acidic system using ChCl / ethylene glycol or ChCl / glycerol in a 1:2 molar ratio, with pH adjusted to 3.5, 4.5, or 5.5 by adding small amounts of imidazole or triethylamine; and a near-neutral system using ChCl / urea in a 1:2 molar ratio, with pH stabilized at 6.0–6.5 by adjusting with trace amounts of acid / base.

[0030] Subsequently, the pectin polysaccharide was added to DES preheated to 60°C and stirred under nitrogen protection for 2–4 h until a clear, homogeneous phase was dissolved. A certain amount of H₂SeO₃ was then added, and the reaction was carried out at 55–65°C for 3–5 h. After the reaction was completed, the mixture was cooled in an ice bath, and three volumes of anhydrous ethanol or acetone were added to precipitate the product. The precipitate was collected by centrifugation, reconstituted with deionized water, and dialyzed against running water for 48 h using a 3500 Da dialysis bag to obtain purified selenized Rhodiola rosea polysaccharide (Se-RRP).

[0031] Example verification: Selenium content and degree of selenium substitution detection The polysaccharide sample (approximately 10 mg) was accurately weighed, microwave-digested with concentrated nitric acid, and then brought to a final volume. The concentration of Se (C, μg / mL) in the digest was determined by ICP-MS. The absolute selenium content was calculated using the formula wSe (mg / g) = C × V / m (where V is the final volume in mL and m is the sample mass in g). The degree of substitution (DS) was then calculated by converting the selenium mass fraction and substituting it into the following formula:

[0032] In the formula, 162 g / mol is the average molecular weight of monosaccharide residues, and 78.96 and 128.97 are the atomic weight of Se and the molecular weight increased by grafting selenite ester group (-SeO2OH), respectively.

[0033] The results of selenium content and degree of substitution (DS) determination showed that different DES systems and apparent pH affected the degree of selenium modification of the product. In the choline chloride / ethylene glycol / imidazole system, the selenium content in the apparent pH 4.5 group was 19.4 ± 3.1 mg / g, and the apparent degree of substitution was 0.041 ± 0.007. The selenium content in the apparent pH 2.5 group was approximately 19.1 mg / g, which was similar to the apparent pH 6.5 group; while the selenium content in the apparent pH 6.5 group was only about 2.2 mg / g. The results indicate that stronger acidity can promote the introduction of selenium-containing components, but may also cause polysaccharide degradation; near-neutral conditions are conducive to structure preservation, but difficult to achieve sufficient modification. The apparent pH 4.5 group showed a better overall balance between selenium content and reaction mildness (see [link to relevant documentation]). Figure 7 , Figure 8 ).

[0034] Molecular weight distribution data Table 1. Molecular weight distribution of selenized Rhodiola rosea polysaccharide products under different DES systems and pH regulation

[0035] The molecular weight distribution of polysaccharides was determined using high performance size exclusion chromatography (HPSEC) combined with a differential refractive index (RI). Samples were prepared as 2.0 mg / mL solutions (mobile phase: 0.1 mol / L NaNO3 solution) and filtered through a 0.22 μm aqueous filter. A TSKgel G5000PWxl gel permeation column was used, maintained at 35℃, with a flow rate of 0.5 mL / min and an injection volume of 20 μL. The Mn, Mw, Mz, and polydispersity index (PDI) of the samples were calculated and output using a standard curve and a chromatography workstation.

[0036] The molecular weight distribution (HPSEC) analysis showed that the original RRP had a weight-average molecular weight of 1,399,133 Da. The weight-average molecular weight decreased to 257,440 Da in the apparent pH 2.5 group, with a molecular weight retention rate of only 18.4%, indicating that strong acid conditions caused significant glycosidic bond breakage and destruction of high molecular weight structures. In the choline chloride / ethylene glycol / imidazole system, the molecular weight retention rates in the apparent pH 3.5, 4.5, and 5.5 groups were 89.9%, 93.8%, and 95.4%, respectively, indicating that the polysaccharide structure retention gradually increased with decreasing acidity. Although the apparent pH 5.5 and 6.5 groups had higher molecular weight retention rates, their selenium content decreased; the apparent pH 4.5 group maintained both a high selenium content and a molecular weight retention rate of 93.8%, which better met the comprehensive screening objectives of this study (see Table 1).

[0037] Glucuronic acid content data The uronic acid assay results showed that the strong acid group had the highest relative uronic acid content. However, considering the significant decrease in its weight-average molecular weight, this increase cannot be explained by better protection of the uronic acid backbone. Stronger acidity may preferentially cause hydrolysis of the side chains and branched structures of neutral sugars such as Ara and Gal, leading to a relative increase in the proportion of GalA in the purified product. Therefore, the increased uronic acid content in the strong acid group is more likely to reflect a change in the original main-chain-side-chain composition ratio. The uronic acid composition of the apparent pH 4.5 group was closer to the original RRP, while maintaining a 93.8% molecular weight retention rate. This indicates that this condition can reduce excessive loss of neutral sugar side chains and degradation of the overall polysaccharide structure, and is more conducive to maintaining the original complex structural characteristics (see [link to relevant documentation]). Figure 9 ).

[0038] Infrared spectroscopy analysis The polysaccharides before and after modification were analyzed by Fourier transform infrared spectroscopy (FT-IR) to confirm the successful grafting of selenium groups. Both the original polysaccharide (RRP) and the selenized polysaccharide (Se-RRP) showed optimal results at 3400 cm⁻¹. -1 and 2930 cm -1 The vicinity exhibits typical -OH and CH stretching vibration peaks of polysaccharides. Compared to RRP, Se-RRP shows peaks at 920–930 cm⁻¹. -1 A strong new absorption peak appeared at 830–850 cm⁻¹, attributed to the asymmetric stretching vibration of Se=O; simultaneously, a strong new absorption peak appeared at 830–850 cm⁻¹. -1 and 670–770 cm -1 The region shows newly added characteristic absorption peaks corresponding to Se-O and CO-Se bonds. Furthermore, the 3400 cm⁻¹ region in Se-RRP exhibits... -1 The change in the -OH absorption peak further indicates that the hydroxyl group in the sugar ring participated in the selenite esterification reaction. These results clearly confirm that the selenite group has been successfully covalently bonded to the polysaccharide backbone (see [link to relevant documentation]). Figure 10 ).

[0039] Ultraviolet spectroscopy analysis The purity of RRP, Se, and Se-RRP was evaluated using a UV-Vis spectrophotometer to assess the polysaccharide purity and selenization grafting effect. The original polysaccharide RRP exhibited typical terminal absorption in the 200–220 nm range, with no significant absorption peaks at 260 nm and 280 nm, indicating high purity and the absence of free nucleic acid and protein impurities. For the selenium source Se, its absorbance was close to zero in the wavelength range above 220 nm. In contrast, the selenized Se-RRP showed a strong and significant new absorption peak at approximately 278 nm, with a significantly enhanced overall absorbance. This new absorption peak is attributed to the formation of the selenite ester group (-O-SeO2H) and its induced n→σ. Electron transition. The above results clearly confirm that selenium has been successfully covalently grafted onto the Rhodiola rosea polysaccharide molecular chain, rather than through simple physical mixing (see [link]). Figure 11 ).

[0040] Solubility Evaluation To evaluate the effect of selenization modification on the physicochemical properties of Rhodiola rosea polysaccharides, the water solubility behavior of RRP and Se-RRP at different concentrations (5, 10, 20, and 30 mg / mL) was compared. The results showed that at a concentration of 5 mg / mL, both RRP and Se-RRP were completely soluble and clear. When the concentration increased to 10 mg / mL, significant undissolved precipitate appeared at the bottom of the RRP centrifuge tube, while Se-RRP remained completely soluble without precipitation, indicating that selenization modification significantly improved the water solubility limit of Rhodiola rosea polysaccharides. With further increases in concentration to 20 mg / mL and 30 mg / mL, both showed precipitation due to supersaturation, but the precipitation volume of Se-RRP was significantly smaller than that of RRP at the corresponding concentration. These phenomena clearly indicate that the covalent grafting of selenite groups (-O-SeO2H) endows the polysaccharide chains with more hydrophilic sites and steric hindrance, effectively weakening the hydrogen bonding aggregation between polysaccharide molecules, thereby significantly improving the water solubility and dispersion stability of Rhodiola rosea polysaccharides at high concentrations (see [link to relevant documentation]). Figure 12 ).

[0041] Saturated solubility detection and solution transmittance detection Saturated solubility: Excess RRP and Se-RRP samples were separately suspended in deionized water and magnetically stirred at 25℃ for 24 h to reach dissolution equilibrium. The suspensions were centrifuged at 10,000 rpm for 15 min to precipitate undissolved matter, and the supernatant was accurately aspirated. The polysaccharide concentration in the supernatant was determined using the phenol-sulfuric acid method, and the absolute saturated solubility (mg / mL) of the sample in water at 25℃ was calculated. The determination was performed in triplicate.

[0042] Solution transmittance detection: RRP and Se-RRP were prepared into a series of concentration gradient solutions of 5, 10, 20, and 30 mg / mL using deionized water, respectively. These solutions were thoroughly ultrasonicated and allowed to stand at room temperature for 30 min. Using deionized water as a blank control, the transmittance (T%) of each sample solution was measured at 600 nm using a UV-Vis spectrophotometer to quantitatively evaluate the clarity and anti-sedimentation stability of the polysaccharide in the aqueous system.

[0043] Table 2: Comparison of saturated solubility of RRP and Se-RRP in water

[0044] Table 3: Comparison of transmittance (T%) of RRP and Se-RRP solutions at different concentration gradients

[0045] The effect of selenization modification on the water solubility of Rhodiola rosea polysaccharides was quantitatively confirmed by absolute saturation solubility and transmittance (T%) tests. The results showed that the absolute saturation solubility of the natural polysaccharide RRP at 25℃ was (8.15±0.32) mg / mL, while the saturation solubility of the selenized polysaccharide Se-RRP significantly increased to (17.68±0.54) mg / mL, an increase of 116.9% (p<0.001). Transmittance data further indicated that at the critical concentration of 10 mg / mL, the transmittance of RRP plummeted to (62.4±1.5)% due to the precipitation of insoluble matter, while Se-RRP maintained a highly clear state of (97.8±0.5)%. This indicates that the covalent grafting of selenite groups endowed the polysaccharide chains with stronger hydrophilicity and electrostatic repulsion, effectively inhibiting intermolecular self-aggregation, thereby significantly improving its water solubility and system dispersion stability (see Tables 2 and 3).

[0046] Micromorphological changes analysis Scanning electron microscopy comparison showed that ( Figure 13 Selenization modification significantly modifies the microstructure of Rhodiola rosea polysaccharides. As shown in Figure X, the unmodified polysaccharide (RRP) exhibits a dense, flat, and continuous bulk structure; however, after modification with the DES homogeneous system, Se-RRP transforms into a coiled, porous, and highly fragmented ribbon structure at low magnification. At ultra-high magnification (60.0 k×), the Se-RRP surface further reveals a loose, porous three-dimensional network self-assembled from a large number of highly dispersed "cauliflower-like" nano-assemblies. This morphological evolution stems from the introduction of selenite groups, which breaks the strong hydrogen bond entanglement between the original polysaccharide molecules. The large selenium-containing groups generate a significant steric hindrance effect, preventing the tight aggregation of polysaccharide chains. This high specific surface area nanoscale porous structure greatly accelerates the penetration and encapsulation of water molecules, perfectly demonstrating at the microscopic conformation level the physicochemical mechanism of the significantly enhanced saturated solubility (increased by 116.9%) and dispersion stability of Se-RRP (see [link to relevant documentation]). Figure 13 ).

[0047] Atomic force microscopy (AFM) 2D and 3D height images revealed significant depolymerization and reconstruction of the molecular spatial conformation of Rhodiola rosea polysaccharide (Se-RRP) after selenization modification using the DES system. Compared to the highly entangled, densely packed, large spherical aggregates (20–60 nm in height) of unmodified raw sugar (RRP), the vertical height of Se-RRP decreased sharply to 2–8.8 nm (maximum only 8.8 nm), with the massive dome-shaped stacking structure completely disappearing and transforming into a highly dispersed branched network and fine particle conformation. This change is attributed to the successful grafting of selenite groups (-O-SeO2H), whose electrostatic repulsion and steric hindrance effectively broke down the hydrogen bond network between the original polysaccharide molecules, inhibiting the disordered entanglement and aggregation of polysaccharide chains. The above comparison clearly confirms that homogeneous selenization modification transforms the polysaccharide chains from a tightly aggregated state to a relaxed branched network structure, strongly demonstrating the improvement in its aqueous dispersibility and solubility at the molecular spatial morphology level (see [link to documentation]). Figure 14 ).

[0048] Dynamic light scattering (DLS) analysis Dynamic light scattering (DLS) measurements reflect the hydrodynamic diameter (Dh) and aggregation state of polysaccharides in aqueous solution. Unmodified polysaccharides (RRP) exhibit a broad particle size distribution in the aqueous phase, with peak values ​​concentrated in the 700–800 nm region, indicating their tendency to form large fluid aggregates through intermolecular hydrogen entanglement. After selenization modification using the DES system, the particle size distribution of Se-RRP shifted towards smaller particle sizes, with peak values ​​decreasing to around 500–600 nm, and the proportion of large aggregates larger than 1000 nm significantly reduced. This change is attributed to the electrostatic repulsion and steric hindrance effects generated by the introduction of selenite groups (-SeO2OH), effectively suppressing molecular entanglement and aggregation in the aqueous phase. The DLS results are highly consistent with the microscopic depolymerization observed by SEM and AFM, confirming that homogeneous selenization modification effectively improves the dispersion state and solution stability of polysaccharides in aqueous systems (see [link to DES]). Figure 15 ).

[0049] Evaluation of in vitro free radical scavenging capacity The direct antioxidant activity of RRP and Se-RRP in cell-free systems was evaluated using ABTS and DPPH free radical scavenging assays. The results showed that both polysaccharides exhibited dose-dependent scavenging abilities against ABTS and DPPH free radicals. In the ABTS system, Se-RRP demonstrated significantly superior scavenging efficiency compared to RRP, achieving 88% scavenging at only 0.05 mg / mL (compared to only 56% for RRP at the same concentration), and reaching a maximum scavenging plateau (close to 100%) at 0.2 mg / mL. In the DPPH system, the IC50 value of Se-RRP was significantly lower than that of the original sugar, achieving a scavenging rate as high as 98% at 0.4 mg / mL, while RRP required 1.0 mg / mL to reach its maximum scavenging response. Therefore, selenization significantly enhances the ability of polysaccharides to donate hydrogen atoms and electrons, greatly improving their chemical antioxidant efficacy in directly scavenging free radicals (see [reference needed]). Figure 16 ).

[0050] Cell compatibility assessment To assess the biocompatibility of RRP and Se-RRP on cardiomyocytes and to determine the safe dosing window for subsequent antioxidant protection experiments, the effects of different concentrations of polysaccharides on the growth of rat cardiomyocytes (H9c2) were detected using the CCK-8 assay.

[0051] The results showed that within the concentration range of 10–250 μg / mL, the relative growth rate of H9c2 cells in both the RRP and Se-RRP groups remained around 1.0, and the cell viability was greater than 95%, indicating that neither polysaccharide had significant cytotoxicity on cardiomyocytes within this dosage range. Notably, when the dosage was further increased to 500–1000 μg / mL, the native polysaccharide RRP showed a significant dose-dependent growth inhibitory effect on H9c2 cells (cell viability decreased to approximately 35% at 1000 μg / mL); while the selenized Se-RRP showed a significant reduction in cytotoxicity, and the cell viability remained above 67% even at a high concentration of 1000 μg / mL, demonstrating significantly better cardioprotective safety and biocompatibility than the native polysaccharide.

[0052] In summary, selenization significantly broadened the safe administration window of the polysaccharide to cardiomyocytes. Therefore, subsequent protective experiments against H2O2-induced oxidative stress damage (ROS clearance and GSH recovery) in H9c2 cardiomyocytes were all conducted within an absolutely safe dosage range (see [link to relevant documentation]). Figure 17 ).

[0053] Intracellular ROS level detection Intracellular ROS content detection by flow cytometry: After drug administration and modeling treatment, cells were collected and resuspended in serum-free medium. DCFH-DA fluorescent probe (final concentration 10 μM) was added, and the cells were incubated at 37°C in the dark for 20 min. Cells were washed three times with serum-free medium to thoroughly remove any probe that had not entered the cells. Finally, the cells were resuspended in PBS, and the fluorescence intensity distribution of each group of cells was detected by flow cytometry in the FITC channel (excitation / emission wavelength 488 / 525 nm). The mean fluorescence intensity (MFI) and the proportion of ROS-positive cells were analyzed.

[0054] The inhibitory effect of polysaccharides on intracellular ROS accumulation was evaluated using the DCFH-DA fluorescent probe combined with flow cytometry. Results showed that the Control group maintained low basal ROS levels; the Model group experienced a burst of intracellular ROS increase under oxidative stress, with a dramatic rightward shift of the fluorescence peak and a high fluorescence positivity rate of 99.5%, indicating that the cells suffered extremely severe oxidative damage. After intervention with ordinary polysaccharide (RRP), the intracellular ROS fluorescence intensity decreased somewhat; however, the selenized polysaccharide (Se-RRP) treatment group exhibited a more significant ROS scavenging ability, with the fluorescence peak shifting significantly to the left (positive rate decreased to 47.4%). These results were highly consistent with intracellular GSH assay data, jointly confirming that selenization modification can significantly enhance the free radical scavenging ability of polysaccharides and effectively alleviate intracellular ROS damage induced by oxidative stress (see [link to relevant documentation]). Figure 18 ).

[0055] Intracellular GSH level detection Experimental Methods: After drug administration and modeling, cells were digested, collected, and washed and resuspended in PBS. A GSH-specific fluorescent probe (e.g., Thiol Tracker Green, final concentration 10 μM) was added, and the cells were incubated at 37°C in the dark for 30 min. After incubation, the cells were washed twice with ice-cold PBS to remove free probes and finally resuspended in 500 μL PBS. Fluorescence signals of each group of cells were acquired using flow cytometry in the FITC channel (excitation / emission wavelengths 488 / 530 nm), and the fluorescence distribution and mean fluorescence intensity (MFI) of GSH in each group of cells were analyzed using FlowJo software.

[0056] Intracellular GSH levels were detected by flow cytometry to assess the antioxidant protective effect of polysaccharides. Results showed that Control group cells exhibited a strong high fluorescence response, indicating that normal cells maintain abundant GSH reserves. In the Model group, fluorescence intensity decreased sharply under oxidative stress (peak shape shifted significantly to the left), indicating that the accumulation of free radicals led to severe depletion of endogenous GSH and an imbalance in cellular redox balance. After intervention with native polysaccharide (RRP), the cellular fluorescence signal moderately recovered; while the fluorescence intensity of the selenized polysaccharide (Se-RRP) treatment group shifted significantly to the right, and its peak shape and proportion of high-fluorescence cells highly overlapped with the Control group. These results confirm that selenization modification significantly enhances the antioxidant activity of polysaccharides, effectively prevents oxidative stress-induced GSH depletion, and significantly restores the cell's endogenous antioxidant defense capacity (see [link to relevant documentation]). Figure 19 ).

[0057] Molecular dynamics simulations and analysis of the binding mechanism with Keap1 Molecular dynamics (MD) simulations and binding free energy calculations: Explicit solvent molecular dynamics simulations of the RRP-Keap1 and Se-RRP-Keap1 complexes were performed using GROMACS software over a period of 100 ns. The protein was subjected to an Amber99sb-ILDN force field, and the ligand parameters for the polysaccharide fragments were obtained from Antemamber and GAFF2 (based on RESP 2 charge distribution). The complexes were placed in a cubic TIP3P water box with a side length of at least 1.0 nm, and an appropriate amount of Na was added. + and Cl - The system charge was neutralized by ions. First, energy minimization was performed over 5000 steps using the steepest descent method; then, constrained equilibration was performed for 100 ps at NVT (308.15 K) and NPT (1 bar) equilibrations, respectively. Finally, the constraints were removed, and a 100 ns unconstrained MD simulation was run while maintaining 308.15 K and 1 bar. RMSD, RMSF, Rg, SASA, and the number of hydrogen bonds were calculated using GROMACS built-in tools (cutoff distance 0.35 nm, cutoff angle 30°). The last 20 ns (200 frames of conformation) of the stable trajectory were extracted, and the binding free energy (ΔG) between the ligand and protein was calculated using g_mmpbsa combined with MM / PBSA (Molecular Mechanics Poisson-Boltzmann Surface Area) method. bind ).

[0058] Molecular dynamics simulations and analysis of the binding mechanism with Keap1: To reveal the interaction mode between selenized polysaccharides and Keap1 protein at the molecular level, 100 ns molecular dynamics (MD) simulations and MM / PBSA binding free energy calculations were performed on the RRP-Keap1 and Se-RRP-Keap1 complexes. Figure 20 ).

[0059] 1. Conformal stability and compactness analysis of the complex (RMSD, Rg & SASA): RMSD: During the 100 ns simulation, the RMSD value of the Se-RRP-Keap1 complex framework converged rapidly and remained stable in the 0.22-0.25 nm range, significantly lower than that of the RRP-Keap1 group (0.25-0.33 nm, with larger fluctuations, Figure A), indicating that selenization modification can significantly enhance the overall conformational stability after the ligand binds to Keap1. Rg and SASA: The results of radius of gyration (Rg) and solvent-accessible surface area (SASA) further confirm that the Rg (2.45-2.50 nm, Figure C) and SASA (130-140 nm^2, Figure D) of the Se-RRP-Keap1 complex are consistently lower than those of the RRP-Keap1 group, indicating that the binding of Se-RRP enables the Keap1 Kelch domain to form a more compact and stable binding pocket with reduced solvent contact.

[0060] 2. Keap1 Key Residue Flexibility (RMSF): The RMSF map (Figure B) shows that the overall flexibility of the Keap1 protein in the residue 300–620 region (Kelch domain) is significantly restricted after Se-RRP binding. Particularly in the three highly flexible loop regions (around residues 380, 460, and 525), the RMSF peak of the Se-RRP group significantly decreased from ~0.33 nm in the protonic group to ~0.23 nm. This indicates that Se-RRP produces a stronger locking effect with these key binding loops, fixing the pocket conformation.

[0061] 3. Hydrogen Bond Network and Binding Free Energy (H-bonds & MM / PBSA): Number of Hydrogen Bonds: Throughout the 100 ns trajectory, the Se-RRP-Keap1 interface maintained a high-density hydrogen bond network (mainly concentrated at 6–9 bonds, peaking at 11), while the RRP-Keap1 group had only 3–6 bonds (Figure E). The selenization-introduced selenium-containing functional groups and their induced electronic effects greatly enriched the donor / acceptor network and strengthened polar interactions. MM / PBSA Free Energy: MM / PBSA calculations show ( Figure XThe binding free energy of Se-RRP to Keap1 is -48.8 kcal / mol, which is significantly better than that of unmodified RRP fragments (-32.5 kcal / mol).

[0062] Simulation results show that selenization modification (Se-RRP) significantly enhances the binding affinity of polysaccharides to Keap1 by increasing interfacial hydrogen bond density, limiting the flexibility of the Keap1 key loop, and driving the complex to shrink toward a more compact conformation. This strong competitive binding mechanism effectively blocks the binding and degradation of Nrf2 by Keap1, thereby activating the Nrf2 antioxidant signaling pathway and providing direct molecular mechanistic support for its aforementioned potent cellular-level antioxidant effects (ROS scavenging and GSH restoration).

[0063] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on its differences from other embodiments. Similar or identical parts between embodiments can be referred to interchangeably. For the apparatus disclosed in the embodiments, since they correspond to the methods disclosed in the embodiments, the description is relatively simple; relevant parts can be referred to the method section.

[0064] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing a structurally preserved side chain selenoized saussurea involucrata pectin polysaccharide, characterized in that, Includes the following steps: S1. Constructing a eutectic solvent system: Mix hydrogen bond acceptors and hydrogen bond donors at a preset molar ratio, heat and stir until clear and transparent to obtain a eutectic solvent system; the apparent pH of the eutectic solvent system is controlled between 3.5 and 5.5; S2. Homogeneous dissolution: Rhodiola polysaccharide is added to the eutectic solvent system preheated to 60°C and stirred for 2-4 hours under nitrogen protection until a transparent homogeneous solution is formed. S3, Selenization reaction: Add selenite to the transparent homogeneous solution obtained in step S2, and react at 55-65℃ for 3-5 hours to obtain the selenization reaction solution. S4. Purification: Cool the selenization reaction solution obtained in step S3 in an ice bath, add a precipitant to precipitate, collect the precipitate by centrifugation, and then purify it by dialysis after redissolving in deionized water to obtain selenized Rhodiola rosea polysaccharide.

2. The production method according to claim 1, characterized by, In step S1, the eutectic solvent system is selected from the choline chloride / ethylene glycol system or the choline chloride / glycerol system.

3. The production method according to claim 2, characterized by, In the choline chloride / ethylene glycol system or the choline chloride / glycerol system, the molar ratio of choline chloride to hydrogen bond donor is 1:2, and the apparent pH is adjusted to 3.5, 4.5 or 5.5 by adding imidazole or triethylamine.

4. The method of claim 1, wherein, In step S1, the apparent pH of the eutectic solvent system is 4.

5.

5. The preparation method according to claim 1, characterized in that, In step S4, the precipitant is anhydrous ethanol or acetone, and the volume added is 3 times the volume of the reaction liquid; the dialysis is performed using a dialysis bag with a molecular weight cutoff of 3500 Da, and dialysis is performed with running water for 48 hours.

6. A structure-preserving side-chain selenized Rhodiola rosea pectin polysaccharide, characterized in that, It is prepared by the preparation method described in any one of claims 1-5.

7. The application of the structure-preserving side-chain selenized Rhodiola rosea pectin polysaccharide as described in claim 6 in the preparation of antioxidant stress agents.

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