Steam-Driven Structural Remodeling of Polygonatum sibiricum Polysaccharides: Correlating Macromolecular Changes with Enhanced Renal Cell Protection

Abstract

The rhizome of Polygonatum sibiricum (PR) is a widely used medicinal and edible herb valued worldwide. Traditional processing by repeated steaming and sun-drying empirically enhances its therapeutic properties, primarily through alterations in its bioactive polysaccharides. This study aimed to elucidate the structure–activity relationship by systematically investigating the structural features and renoprotective activities of polysaccharides from PR and processed PR (PPR). Polysaccharides from PR and PPR (designated PRP and PPRP, respectively) were isolated and identified as five fractions (PRP-1, PRP-2, PPRP-1, PPRP-2, and PPRP-3). PPRP exhibited a broader molecular weight distribution (1.9–21.5 kDa) compared to PRP (2.6 kDa), indicating degradation and repolymerization. 1D/2D NMR analysis revealed that PRP-1 contained a backbone of →1)-β-D-Fruf-(2→, while processing introduced an additional →5)-α-L-Araf-(1→ linkage into PPRP-1. The acidic fractions, PPRP-2 and PPRP-3, showed a partial overlap of motifs, including →4)-α-D-GalAp-(1→ (in both methylated and non-methylated forms), →4)-β-D-Galp-(1→, →5)-α-L-Araf-(1→, and →2,4)-α-L-Rhap-(1→. Notably, PPRP-2 demonstrated superior renoprotective activity in high glucose-induced NRK-52E cells. These structural transformations provide a molecular basis for the enhanced bioactivity and functional-food potential of polysaccharides.

1. Introduction

The rhizome of Polygonatum sibiricum (PR), a variety of “Huangjing”, is documented in the pharmacopeia [1]. With a history of use spanning over a millennium, it plays a significant role in both Chinese culinary and medicinal customs. The primary avenues for the development of PR-based functional foods include beverages, alcoholic products, tea-based preparations, and prepared instant herbal slices [2]. In Traditional Chinese Medicine (TCM), it is widely recognized for its abilities to replenish qi and yin, tonify the spleen, moisten the lungs, and enhance kidney function, establishing its status as a valuable herbal remedy. Huangjing contains various ingredients, including polysaccharides, saponins, and flavonoids [3]. Recent pharmacological studies have demonstrated that Huangjing ameliorates diabetes and its complications. These metabolic disturbances are key drivers of renal injury, with oxidative stress and the associated inflammatory response constituting a pivotal link in this pathology. Therefore, the antioxidant capacity of Huangjing polysaccharides is recognized as the key mechanism mediating these renoprotective effects [4].

Diabetic kidney disease (DKD) has become the primary cause of end-stage renal disease (ESRD) worldwide [5]. A key mechanism driving its progression is sustained hyperglycemia, which directly induces oxidative stress and injury to renal tubular epithelial cells [6]. Although current standard therapies, including renin-angiotensin system (RAS) inhibitors and sodium-glucose cotransporter-2 (SGLT2) inhibitors, can delay the progression of DKD by modulating hemodynamics and metabolism, they frequently offer inadequate direct cytoprotection against the underlying cellular oxidative damage in these cells [7,8]. Therefore, developing safe, natural compounds with renoprotective properties represents a promising therapeutic strategy.

In both nutraceuticals and clinical applications, processed PR (PPR) is the preferred choice over raw PR, as the latter can cause throat irritation owing to the presence of needle-shaped calcium oxalate crystals [9]. In addition to eliminating this irritation, steaming enhances various bioactivities of Huangjing polysaccharides, such as immunomodulatory and antioxidant effects [10,11]. This enhancement stems from alterations in polysaccharide structure, including changes in molecular weight (degradation or aggregation) and monosaccharide composition. This efficacy-boosting steaming method is also widely applied to other medicinal herbs, such as Rehmannia glutinosa and Panax ginseng [12,13], demonstrating its broad applicability. As a physical processing method that does not introduce exogenous chemical reagents or enzymes, steam treatment conforms to the principles of green and sustainable production for functional foods. However, further research is required to comprehensively elucidate the effects of steaming on the structure and bioactivity of Huangjing polysaccharides. The polysaccharides in raw PR (PRP) are primarily composed of neutral fractions consisting of fructose (Fru) and glucose (Glc) [14,15,16]. Although one study investigated the effects of processing on the polysaccharides of P. kingianum, it focused exclusively on neutral fractions [17]. Consequently, comparative studies are still lacking regarding the full spectrum of polysaccharides (ranging from neutral to acidic) obtained via stepwise elution, particularly regarding their structural evolution and bioactivity changes before and after steaming.

Therefore, the objective of this study is to systematically compare the structural differences between PRP and PPR polysaccharides (PPRP) and to evaluate their protective effects on NRK-52E cells. To this end, five purified fractions, PRP-1, PRP-2, PPRP-1, PPRP-2, and PPRP-3, were obtained via DEAE-cellulose chromatography. The investigation adopted a comprehensive methodology covering three principal aspects: structural elucidation through analyses of molecular weight (Mw), Fourier transform infrared spectroscopy (FT-IR), monosaccharide composition, methylation, and NMR; physicochemical characterization, which included chromaticity, microscopic morphology, and thermal stability; and an evaluation of renoprotective activity against high glucose-induced injury. Ultimately, this work seeks to decipher the molecular basis of the traditional processing method, translating empirical wisdom into a scientific basis for modern nutraceutical development.

2. Materials and Methods

2.1. Materials and Reagents

Fresh rhizomes of P. sibiricum. were sourced from Qingyuan, Liaoning Province, and identified by Prof. Yingni Pan. PR and PPR were prepared according to a previously described method [18]. DEAE-cellulose-52 was obtained from Phygene Life Sciences Co., Ltd. (Fuzhou, China). The monosaccharide standards (purity ≥ 98%) were purchased from Sigma-Aldrich Trading Co., Ltd. (Shanghai, China). Trifluoroacetic acid (TFA) was purchased from ANPEL Laboratory Technologies, Inc. (Shanghai, China). Dimethyl sulfoxide (DMSO) was purchased from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China) Iodomethane was purchased from Meryer Chemical Technology Co., Ltd. (Shanghai, China). Fetal bovine serum (FBS) was obtained from Cellmax (Lanzhou, China). Cell Counting Kit-8 (CCK 8), Dulbecco’s modified Eagle’s medium (DMEM) (high and low glucose), and BCA kits were obtained from Meilun Biotechnology Co., Ltd. (Dalian, China). Metformin HCl (Met) was obtained from Selleck Chemicals (Houston, TX, USA). SOD, MDA, and GSH were sourced from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China), and ELISA kits for fibronectin (FN), collagen type I (COL1), and α-smooth muscle actin (α-SMA) were obtained from Shanghai Jianglai Biotechnology Co., Ltd. (Shanghai, China). All other chemicals and reagents were of analytical grade.

2.2. Extraction of Crude Polysaccharides

PR and PPR decoction pieces (100 g) were defatted with ethanol, and then extracted twice by reflux for 1 h each, with 9- and 7-times the volume of water, respectively. The combined filtrates were concentrated at 50 °C under reduced pressure, respectively. After removing protein using Sevag reagent (dichloromethane: n-butanol = 4:1, v/v), 4 volumes of anhydrous ethanol were added to precipitate the mixture overnight, followed by lyophilization to obtain crude polysaccharides.

2.3. Isolation and Purification of PRP and PPRP

The PRP and PPRP were dissolved in ultrapure water and centrifuged. The supernatant was applied to a DEAE-52 cellulose column (2.5 × 40 cm) and eluted sequentially with pure water, 0.05 M, 0.1 M, 0.2 M, 0.3 M, 0.4 M, and 0.5 M sodium chloride solution. Each fraction collected was 10 mL, and 3 column volumes were eluted for each gradient. Absorbance (582 nm) was determined every 5 tubes via anthrone-sulfuric assay [19]. The fractions exhibited identical absorption peaks were pooled, concentrated, dialyzed, and subsequently freeze-dried. Two polysaccharide fractions were obtained from PRP, designated PRP-1 and PRP-2, and the other three polysaccharide fractions obtained from PPR were designated PPRP-1, PPRP-2, and PPRP-3.

2.4. Structural Analysis of the Polysaccharides

2.4.1. UV Spectroscopy

Five polysaccharides were dissolved in ultrapure water (1 mg/mL), respectively, with the water used as a blank solvent, and full wavelength UV scanning was conducted over a 200–400 nm range using a UV-2600i spectrophotometer (Shimadzu Corporation, Kyoto, Japan) with a quartz cuvette.

2.4.2. Purity and Mw Analysis

Polysaccharide solutions (5 mg/mL) and dextran standard solutions were filtered (0.22 μm) and analyzed by high-performance gel permeation chromatography (HPGPC) on a Thermo U3000 system equipped with RID detector (Thermo Fisher Scientific, Waltham, MA, USA) and a BRT105-103-101 column (BoRui Saccharide Biotech Co., Ltd., Yangzhou, China) at 40 °C (0.7 mL/min, ultrapure water eluent). The molecular weights were determined using dextran standards (5.8–853 kDa).

2.4.3. Determination of the Sugar, Uronic Acid, and Protein Contents

The total carbohydrate content was determined by using the anthrone-sulfuric method, with glucose (Glc) as the standard. The uronic acid content was determined by using the meta-hydroxybiphenyl method, and the galacturonic acid (GalA) was used as a standard [20]. The protein content was measured by using the bicinchoninic acid method, with bovine serum albumin (BSA) as the standard.

2.4.4. FT-IR Spectroscopy

Each dried polysaccharide sample (1 mg) was mixed with KBr and pressed into transparent pellets. A Fourier-transform infrared spectrometer (FT-IR 650, Tianjin Gangdong Sci. and Tech. Co., Ltd., Tianjin, China) was used to record infrared spectra in the range of 400–4000 cm⁻¹.

2.4.5. Monosaccharide Composition Analysis

The monosaccharide composition was determined by ion chromatography (IC) using a ICS5000+ system equipped with a CarboPac PA20 column (150 × 3.0 mm, 10 μm) (Thermo Fisher Scientific) at 30 °C [21]. Fru is heat-sensitive and degrades during prolonged high-temperature processing, making its quantification challenging. To address this, two distinct hydrolysis methods were used for monosaccharide composition analysis [14]. PRP-1, PRP-2, and PPRP-1 were subjected to partial acid hydrolysis (60 °C, 1 h), whereas PPRP-2 and PPRP-3 underwent complete hydrolysis (121 °C, 2 h). The samples were then dried under nitrogen, methanol-washed (2–3 repeats), redissolved in ultrapure water, and analyzed. A working standard mixture (10 μg/mL) was prepared by combining individual monosaccharide stock solutions (10 mg/mL) for quantification.

2.4.6. Congo Red Analysis

Congo red complexes with helical polysaccharides, inducing a red shift in the maximum absorption wavelength (λmax) that reflects the conformation of the polysaccharide. Briefly, 2.0 mL of polysaccharide solutions (0.5 mg/mL) were mixed with 2.0 mL of Congo Red (100 μM) and scanned from 400 to 800 nm, varying NaOH gradients (0–0.6 M, 4 mL). Control contained Congo Red alone.

2.4.7. Methylation and GC-MS Analysis

To elucidate the polysaccharide structure, the glycosidic linkages of the polysaccharides were analyzed by methylation and GC-MS [22]. Acidic polysaccharides were first reduced prior to methylation. Briefly, 30 mg of reduced polysaccharide samples (PRP-1 and PPRP-1) were dissolved in 10 mL of DMSO, then stirred for 2 h at room temperature after adding 30 mg of NaOH powder. The reaction mixture was cooled in an ice bath, and 4 mL of iodomethane was added dropwise with stirring in the dark for 2 h. The reaction was quenched by adding 1 mL of water. The methylated polysaccharides were extracted with CH₂Cl₂, washed several times with distilled water, and dried over an anhydrous Na₂SO₄ column. The methylated samples were subjected to hydrolysis with formic acid hydrolysis, reduction with NaBH₄, and acetylation with acetic anhydride before being redissolved in 2.0 mL of CHCl₃ for partially methylated alditol acetate (PMAA) analysis by GC-MS.

PMAA derivatives were analyzed by GC-MS (GCMS-TQ 8040, Shimadzu) using an HP-5 MS column (30 m × 0.25 mm, 0.25 μm; Agilent, Santa Clara, CA, USA). Injection port: 250 °C. Temperature program: 0–2 min at 80 °C; 2–5 min with a ramp from 80 °C to 140 °C; 5–6 min held at 140 °C; 6–13.5 min with a ramp from 140 °C to 155 °C; 13.5–32.5 min with a ramp from 155 °C to 250 °C; and finally, 32.5–37.5 min held at 250 °C.

2.4.8. NMR Spectroscopy

Each dried polysaccharide sample (30 mg) was dissolved in 0.55 mL D₂O, repeatedly freeze-dried, and dissolved in 0.55 mL D₂O. The ¹H NMR, ¹³C NMR, DEPT-135, COSY, HMBC, HSQC, and NOESY were obtained using a 600 MHz Bruker AVANCE III HD spectrometer (Bruker BioSpin GmbH, Karlsruhe, Germany).

2.5. Physicochemical Property Analysis

2.5.1. Chromaticity and CIE- L*a*b*Analysis

Chromaticity analysis was conducted in accordance with the Chinese Pharmacopeia. Color measurement and quantification were performed using the CIE L*a*b* color system with a precision colorimeter (3nh, Shenzhen, China), and ultrapure water serving as the control [23]. The total color difference (ΔE*) value was calculated according to Equation (1).

$${∆E}^{*}=\sqrt{{∆L}^{2 }+∆a^2+{∆b}^2}$$ (1)

2.5.2. Scanning Electron Microscope (SEM) Analysis

The freeze-dried polysaccharides were mounted on stages with conductive adhesive, sputter-coated with gold using a Hitachi MC1000 ion sputter coater (Hitachi, Tokyo, Japan) for approximately 30 s, and examined by SEM (Hitachi SU8600) at 3.0 kV to characterize the morphology of the five samples.

2.5.3. Thermal Stability Analysis

Thermal stability analysis of five polysaccharide samples was performed with a thermogravimetric differential scanning calorimetry analyzer (TG-DSC, PerkinElmer STA 8000, Waltham, MA, USA). Briefly, 8 mg of the sample was placed into a crucible with a temperature ramp (30–800 °C, 10 °C/min) under N₂ atmosphere [24].

2.6. Evaluation of the Renal Protective Effects on NRK-52E Cells

2.6.1. Cell Culture

NRK-52E cells were obtained from Servicebio Technology Co., Ltd. (Wuhan, China; Catalog Number: STCC30003P), and were cultured in DMEM supplemented with 5% FBS under normal glucose (NG, 5.5 mM glucose) or high glucose (HG, 30 mM glucose) [25]. The cells representing each condition were seeded into 75 cm² flasks and incubated at 37 °C with 5% CO₂ for 48 h.

2.6.2. Cell Viability Assay

NRK-52E cells (100 μL, 1.0 × 10⁵ cells/mL) were plated in a 96-well plate and cultured for 6 h. The medium was replaced with fresh medium devoid of FBS but supplemented with various polysaccharides (12.5, 25, 50, and 100 μg/mL) or Met (5 μg/mL). After 48 h of incubation, the old medium was exchanged for 100 μL medium containing 10% CCK-8, and then incubated for 2 h before measuring absorbance at 450 nm (Varioskan Lux, Thermo). Cell viability was calculated using Equation (2):

$$\mathrm{C}\mathrm{e}\mathrm{l}\mathrm{l} \mathrm{v}\mathrm{i}\mathrm{a}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}\left(\mathrm{\%}\right)=A_{\mathrm{T}}/A_{\mathrm{C}}\times 100\mathrm{\%}$$ (2)

where A_T and A_C, respectively, represent absorbance values of the test and control groups.

2.6.3. Measurement of Oxidative Stress Markers (SOD, GSH, MDA)

Cells were lysed and centrifuged (10,000× g, 10 min, 4 °C) to collect the supernatant. The protein concentration in the supernatant was quantified using the BCA assay. The supernatant was then used for all subsequent biochemical analyses.

SOD activity: SOD activity was determined using the water-soluble tetrazolium salt (WST-1) method. A 20 µL sample was mixed with 20 µL of enzyme working solution and 200 µL of WST-1 substrate. Following incubation at 37 °C for 20 min, absorbance was measured at 450 nm.

GSH content: GSH levels were measured by the 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) colorimetric method. A 100 µL sample was mixed with 25 µL of chromogenic reagent and 100 µL of buffer. After incubation at room temperature for 5 min, absorbance was read at 405 nm.

MDA content: MDA levels were assessed using the thiobarbituric acid (TBA) method. A 100 µL sample was mixed sequentially with 100 µL of clarifying reagent, 1.5 mL of working solution, and 1.5 mL of color-developing reagent. The mixture was incubated at 95 °C for 40 min, cooled, and centrifuged (10,000× g, 10 min). The absorbance of the supernatant was measured at 532 nm.

All results were normalized to protein concentration and expressed per mg protein.

2.6.4. Measurement of Fibrosis-Related Proteins (FN, COL1A, α-SMA)

Cell culture supernatants were harvested and centrifuged (1000× g, 20 min, 4 °C). The clarified supernatant was analyzed directly. All incubations were performed at 37 °C. Briefly, 100 µL of standard or sample was added to each well and incubated for 60 min. After aspiration, 100 µL of biotinylated detection antibody was added to each well and incubated for 60 min. The wells were washed three times (300 µL/well) and blotted dry. Subsequently, 100 µL of HRP-conjugated streptavidin was added to each well and incubated for 30 min. After five washes, 90 µL of TMB substrate was added and incubated for 15 min in the dark. The reaction was stopped with 50 µL of stop solution, and absorbance was immediately measured at 450 nm.

2.7. Statistical Analysis

Data were expressed as mean ± SD. Due to violations of normality (Shapiro–Wilk test) and homogeneity of variances (Levene’s test), the Kruskal–Wallis H test was applied for group comparisons, with Dunn’s test (Holm–Bonferroni corrected) used for post hoc analysis following a significant result. Statistical analysis and graphing were performed using Origin (v 2024) and GraphPad Prism (v 8.0), while MestReNova (v 14.0) was utilized for the NMR spectra analysis.

3. Results and Discussion

3.1. Polysaccharide Isolation and Purification

After water extraction, deproteinization, and alcohol precipitation, the yield of PRP and PPRP was 15.21% and 3.95%, respectively (Figure 1 and Figure 2). PRP and PPRP appeared as white and brownish-black powders, respectively. The two sub-fractions from PRP were designated PRP-1 and PRP-2, and three sub-fractions from PPRP were designated PPRP-1, PPRP-2, and PPRP-3. There was a significant decrease in the proportion of neutral polysaccharides and a corresponding increase in that of acidic ones following steaming.

Figure 1.

Flow chart for the separation and purification of PRP and PPRP.

Figure 2.

Stepwise elution curve of PRP and PPRP.

3.2. Structural Characterization of the Polysaccharides

3.2.1. UV Analysis

UV spectroscopy is commonly employed to preliminarily assess the purity of polysaccharides by detecting potential impurities, such as proteins (280 nm) and nucleic acids (260 nm) [26]. The result revealed low protein and nucleic acid contents in the five polysaccharides (Figure S1).

3.2.2. Mw and Chemical Composition Analysis

The purity and molecular weight distribution of polysaccharides are critical factors influencing their bioactivity. Therefore, the HPGPC chromatograms for the five polysaccharides displayed a single symmetrical peak (Figure S2), showing that they were homogeneous. Mw analysis revealed determinations showed a decrease for PPRP-1 but an increase for PPRP-2 and PPRP-3 compared with PRP-1 and PRP-2. These divergent trends suggested that processing induced the degradation and subsequent reaggregation of the polysaccharides. The Mw values, along with the contents of total polysaccharides, uronic acid, and protein, are summarized in Table 1.

Table 1.

Molecular weights and contents of total polysaccharides, uronic acid, and protein from PRP and PPRP.

	PRP-1	PRP-2	PPRP-1	PPRP-2	PPRP-3
Mw (kDa)	2.619	2.608	1.894	11.084	21.499
Polysaccharide (%)	88.93	72.58	84.96	87.70	85.77
Uronic acid (%)	5.67	7.96	6.96	62.33	66.26
Protein (%)	2.97	1.78	1.06	4.74	3.88

3.2.3. FT-IR Analysis

FT-IR spectroscopy is used to identify characteristic functional groups, determine the configuration of the glycosidic linkages, and identify the type of sugar ring [27,28]. A broad and strong absorption band at 3400 cm⁻¹ was observed, corresponding to O-H stretching vibrations, accompanied by deformation modes at 1400 cm⁻¹. The peak at 2930 cm⁻¹ was attributed to C-H stretching vibrations. The absorption peak at 1640 cm⁻¹ was assigned to C=O stretching vibration (carboxyl group) or the presence of bound water. Additionally, the absorption peak at 1020 cm⁻¹ was attributed to the stretching vibration of the C-O-C glycosidic bonds in the sugar ring [28]. In addition, PPRP-2 and PPRP-3 exhibited distinct carboxyl vibrational peaks at 1740 cm⁻¹, coupled with increased galacturonic acid content, indicating significant acetylation or esterification. Specifically, the degree of esterification (DE) was determined to be 0.76 and 0.92 for PPRP-2 and PPRP-3, respectively. Concomitant with this structural shift, the characteristic β-D-furan fructose peaks (933 and 809 cm⁻¹) were absent, providing direct structural evidence for the fructose degradation observed in monosaccharide analysis [29]. The results were shown in Figure 3A.

Figure 3.

(A) The FT-IR spectra of polysaccharides. (B) The chromatograms of the monosaccharide composition of polysaccharides.

3.2.4. Monosaccharide Composition Determination

The monosaccharide composition was analyzed to identify the constituent sugar units of the polysaccharides. IC was utilized to analyze the monosaccharide compositions of the five polysaccharides. The compositional analysis indicated that PRP-1 and PRP-2 consisted of Fru and Glc, and PPRP-1 was composed of Fru, Glc, and arabinose (Ara). The concentrations of Glc and Fru in PPRP were reduced or even absent. Interestingly, the monosaccharide profiles of PPRP-2 and PPRP-3 were primarily composed of Ara, rhamnose (Rha), galactose (Gal), and GalA. The fructan structure is unstable, with its backbone β-(2→1) bonds being highly susceptible to hydrolysis under thermal or humid-heat environments [30]. Prolonged exposure to high-temperature steaming resulted in extensive hydrolysis of fructans, leading to the cleavage of molecular chains and the production of significant quantities of fructooligosaccharides, sucrose, and ultimately, free fructose. The accumulation of these low-molecular-weight sugars likely contributes to the characteristic sweetness of PPR. Simultaneously, the thermal and hydrolytic processes during treatment progressively compromised the cell wall architecture, disrupting the cellulose-pectin-hemicellulose complex. This breakdown liberated previously encapsulated, insoluble pectic polysaccharides, facilitating their subsequent dissolution [31,32]. The specific molar ratios are shown in Figure 3B and Table 2, indicating that the processing of PR into PPR resulted in significant alterations in polysaccharide composition.

Table 2.

Monosaccharide compositions of PRP and PPRP.

Sample	Molar Ratio (%)
	Rha	Ara	Gal	Glc	Fru	GalA
PRP-1	-	-	-	5.52	94.48	-
PRP-2	-	-	-	5.47	94.53	-
PPRP-1	-	0.41	-	6.64	92.95	-
PPRP-2	8.68	15.31	57.61	0.60	-	17.80
PPRP-3	15.03	12.08	48.65	-	-	24.25

3.2.5. Congo Red Examination

The Congo red analysis results were presented in Figure S3. Relative to the Congo Red blank, the λmax of PPRP-3 exhibited a red shift within 0 and 0.1 mol/L, but subsequently decreased with increasing alkaline concentration. This indicated a weak triple helix structure. In contrast, the other polysaccharides did not show a triple helix structure. The underlying mechanism for this disparity appears to be molecular weight-dependent. The lower molecular weights of the other fractions (1.9–11 kDa) fall short of the critical chain length necessary to initiate and stabilize helical folding. Conversely, the higher molecular weight of PPRP-3 (21.5 kDa) exceeds this fundamental threshold, permitting the formation of a weak and unstable triple-helix structure [33].

3.2.6. Methylation Analysis

Results from the methylation analysis of the polysaccharides were compiled in Table 3. During derivatization, the reduction in the carbonyl group at the C-2 position of fructose yields glucitol and mannitol, which were subsequently acetylated to form a mixture of their respective acetate derivatives [34]. According to the data of monosaccharide composition, the sugar residues of PRP-1, PRP-2, and PPRP-1 included →6)-Fruf-(2→, →1,6)-Fruf-(2→), Fruf-(2→, →1)-Fruf-(2→ and →6)-Glcp-(1→; PPRP-2 included Araf-(1→, →1)-Araf-(5→, →3,5)-Araf-(1→,→2,4)-Rhap-(1→, Galp-(1→, →4)-Galp-(1→, →4)-Glcp-(1→, →4,6)-Galp-(1→, no longer contained fructose. PPRP-3 contained →2,3,5)-Araf-(1→, →2,4)-Rhap-(1→, →4)-Galp-(1→, →6)-Galp-(1→, →4,6)-Galp-(1→, →3,6)-Galp-(1→, no longer contained glucose.

Table 3.

Linkage patterns and methylation status of polysaccharides.

Polysaccharide	RT (min)	PMAA	Diagnostic Fragments	Deduced Resides
PRP-1	21.767	2, 5-di-O-acetyl-1, 3, 4, 6-tetra-O-methyl hexitol mannitol	71, 75, 87, 101, 129, 145, 161, 205, 245	Fruf-(2→
	22.081	2, 5-di-O-acetyl-1, 3, 4, 6-tetra-O-methyl hexitol glucitol	71, 75, 87, 101–102, 129, 145, 161–162, 205, 245	Fruf-(2→
	26.343	2, 5, 6-tri-O-acetyl-1, 3, 4-tri-O-methyl mannitol	71, 75, 87, 101, 129, 145, 161, 189, 233	→6)-Fruf-(2→
	26.910	1, 2, 5-tri-O-acetyl-3, 4, 6-tri-O-methyl mannitol	71, 87, 99, 101, 129–130, 145, 161–162, 189, 205, 233	→1)-Fruf-(2→
	28.318	1, 5, 6-tri-O-acetyl-2, 3, 4-tri-O-methyl glucitol	71, 87, 99, 101, 117, 129–130, 161, 189, 203, 233	→6)-Glcp-(1→
	31.305	1, 2, 5, 6-tetra -O-acetyl-3, 4-di-O-methyl mannitol	71, 87, 99, 129, 189, 203, 233	→1, 6)-Fruf-(2→
PRP-2	21.811	2, 5-di-O-acetyl-1, 3, 4, 6-tetra-O-methyl hexitol mannitol	71, 75, 87, 101, 129, 145, 161, 205, 245	Fruf-(2→
	26.947	1, 2, 5-tri-O-acetyl-3, 4, 6-tri-O-methyl mannitol	71, 87, 99, 101, 129–130, 145, 161–162, 189, 233	→1)-Fruf-(2→
	27.130	1, 2, 5-tri-O-acetyl-3, 4, 6-tri-O-methyl glucitol	71, 87, 99, 101, 129–130, 145, 161–162, 189	→1)-Fruf-(2→
	28.305	1, 5, 6-tri-O-acetyl-2, 3, 4-tri-O-methyl glucitol	71, 87, 99, 101, 117, 129–130, 161, 189, 203, 233	→6)-Glcp-(1→
	31.324	1, 2, 5, 6-tetra -O-acetyl-3, 4-di-O-methyl mannitol	71, 87, 99, 129, 189, 203, 233	→1, 6)-Fruf-(2→
PPRP-1	21.765	2, 5-di-O-acetyl-1, 3, 4, 6-tetra-O-methyl hexitol mannitol	71, 75, 87, 101, 129, 145, 161, 205, 245	Fruf-(2→
	26.900	1, 2, 5-tri-O-acetyl-3, 4, 6-tri-O-methyl mannitol	71, 87, 99, 101, 129–130, 145, 161–162, 189, 233	→1)-Fruf-(2→
	27.085	1, 2, 5-tri-O-acetyl-3, 4, 6-tri-O-methyl glucitol	71, 87, 99, 101, 129–130, 145, 161–162, 189, 233	→1)-Fruf-(2→
	28.275	1, 5, 6-tri-O-acetyl-2, 3, 4-tri-O-methyl glucitol	71, 87, 99, 101, 117, 129–130, 161, 189, 203, 233	→6)-Glcp-(1→
	31.295	1, 2, 5, 6-tetra-O-acetyl-3, 4-di-O-methyl mannitol	71, 87, 99, 129, 189, 203, 233	→1, 6)-Fruf-(2→
PPRP-2	17.074	1, 4-di-O-acetyl-2, 3, 5-tri-O-methyl arabinitol	71, 87, 99, 101, 117, 129, 145, 161	Araf-(1→
	21.865	1, 3, 4-tri-O-acetyl-2, 5-di-O-methyl arabinitol	71, 87, 99, 102, 118, 129, 189	→1)-Araf-(5→
	24.585	1, 5-di-O-acetyl-2, 3, 4, 6-tetra-O-methyl galactitol	71, 87, 99, 101, 117, 129, 145, 161, 205	Galp-(1→
	25.228	1, 3, 4-tri-O-acetyl-2, 5-di-O-methyl arabinitol	73, 99, 117, 127, 159, 261	→3, 5)-Araf-(1→
	25.775	1, 2, 4, 5-tetra-O-acetyl-6-deoxy-3-O-methyl rhamnitol	74, 87, 101, 117, 129, 143, 159, 189, 203	→2, 4)-Rhap-(1→
	27.503	1, 4, 5-tri-O-acetyl-2, 3, 6-tri-O-methyl galactitol	71, 87, 99, 101, 117, 129, 131, 175, 203, 233	→4)-Galp-(1→
	27.767	1, 4, 5-tri-O-acetyl-2, 3, 6-tri-O-methyl glucitol	71, 87, 101, 99, 101, 113, 117, 131, 161, 173, 233	→4)-Glcp-(1→
	31.442	1, 4, 5, 6-tetra-O-acetyl-2, 3-di-O-methyl galactitol	85, 99, 101, 117, 127, 141, 159, 201, 231, 261	→4, 6)-Galp-(1→
PPRP-3	25.759	1, 2, 4, 5-tetra-O-acetyl-6-deoxy-3-O-methyl rhamnitol	74, 87, 101, 117, 129, 143, 159, 189, 203	→2, 4)-Rhap-(1→
	27.475	1, 4, 5-tri-O-acetyl-2, 3, 6-tri-O-methyl galactitol	71, 87, 99, 101, 117, 129, 131, 173, 203, 233	→4)-Galp-(1→
	28.278	1, 5, 6-tri-O-acetyl-2, 3, 4-tri-O-methyl galactitol	71, 87, 99, 101, 117, 129, 161, 173, 189, 233	→6)-Galp-(1→
	31.418	1, 4, 5, 6-tetra-O-acetyl-2, 3-di-O-methyl galactitol	85, 99, 101, 117, 127, 141, 159, 201, 231, 261	→4, 6)-Galp-(1→
	32.210	1, 3, 5, 6-tetra-O-acetyl-2, 4-di-O-methyl galactitol	87, 99, 101, 117, 129, 139, 159, 189, 201, 233	→3, 6)-Galp-(1→
	34.935	1, 2, 3, 4, 5-penta-O-acetyl arabinitol	73, 97, 103, 115, 127, 139, 157, 170, 187, 217	→2, 3, 5)-Araf-(1→

3.2.7. NMR Analysis

1D/2D NMR spectroscopy provided definitive evidence for the glycosidic linkage types and anomeric configurations. The primary structures of several polysaccharides were elucidated through comprehensive NMR analysis, including ¹H NMR, ¹³C NMR, DEPT-135, as well as 2D experiments (COSY, HSQC, HMBC, and NOESY). The ¹H and ¹³C NMR spectral data are presented in Table S1. The structural characterization results indicated that PRP-1, PRP-2, and PPRP-1 contained fructans, whereas PPRP-2 and PPRP-3 were identified as pectic galactans. Comprehensive structural elucidation was subsequently conducted on PRP-1 and PPRP-2, focusing on their glycosidic linkage patterns and monosaccharide compositions.

Chemical shift assignment of PRP-1

The ¹H NMR spectrum of PRP-1 (Figure 4A) revealed that the proton signals were predominantly distributed between δ 3.5 and 5.5. The δ 5.0–5.5 and δ 4.4–5.0 ranges served as diagnostic markers for α distinguishing. β glycosidic linkages [35], with the signal at δ 5.41 indicating an α-configuration sugar residue. In conjunction with ¹³C NMR (Figure 4B) and the two-dimensional spectra, the terminal signals at δ 5.41/92.45 were connected sequentially to δ 3.55/71.41, 3.77/72.62, 3.48/69.37, 3.96/71.77, and 3.71/63.29, which suggested the presence of →6)-α-D-Glcp-(1→(F) [36]. The ¹³C NMR spectrum showed five sets of terminal signals, except at δ 92.45. The remaining four signals were observed at δ 104.42, 104.02, 103.88, and 103.40, all indicative of β-configurations and were absent from the DEPT-135 spectrum (Figure 4C), which indicated that these signals were quaternary carbons, characteristic of fructose C-2 signals. These results indicated that fructose and glucose were the dominant constituents of PRP-1. Specifically, the signal at δ 103.40 corresponded to the C-2 signal of →1)-β-D-Fruf-(2→, whereas δ 103.88, 104.02, and 104.42 represented C-2 of β-D-Fruf-(2→, →1,6)-β-D-Fruf-(2→, and →6)-β-D-Fruf-(2→, respectively [14]. In the ¹³C NMR spectrum, the methylene carbon signals in the high-field region (δ 60.60–63.54) were attributed to the C-1 and C-6 signals from the fructose residues. The C-5 signal of the fructose residues was observed at δ 81.19 and 80.21, the C-4 signal at δ 74.83, and the C-3 signal at δ 76.79 and 77.45. By comparison with the literature data, these four sets of signals were assigned to →6)-β-D-Fruf-(2→ (A), →1,6)-β-D-Fruf-(2→(B), β-D-Fruf-(2→(D), and →1)-β-D-Fruf-(2→(E). HMBC and NOESY spectra were employed to further analyze the glycosidic linkages and sequences between sugar residues [37]. Specifically, the cross-peaks in the HMBC spectrum at δ 5.43/103.40 and 3.71/103.88 indicated linkages between the H-1 of the sugar residue F and the C-2 of E, and between the H-1 of F and the C-2 of D. Furthermore, correlations at δ 3.66/104.42, 3.66/103.88, and 3.89/103.40 suggested possible linkages between the H-6 of B and the C-2 of A, the H-6 of A (B H-6) and the C-2 of D, and the H-1 of B and the C-2 of E. By integrating the proton signals of the terminal hydrogen of Glcp and the H-3 of the Fruf sugar residues in the ¹H NMR spectrum, the ratio of Glcp to the Fruf residues was determined to be 1:11. In the ¹³C NMR spectrum, the ratio of the C-2 signals of the Fruf residues is A:B:D:E = 1:2:3:5, indicating that PRP-1 is likely a fructan with an average degree of polymerisation of 12. It consisted primarily a →1)-β-D-Fruf-(2→ backbone. The proposed structure is shown in Figure 4H. The structures of PRP-2 and PPRP-1 were also elucidated (Figures S4 and S5). PRP-2 and PPRP-1 showed structural similarities to PRP-1. Notably, PPRP-1 contained an additional →5)-α-L-Araf-(1→(G) linkage compared with PRP-1 and PRP-2.

Figure 4.

NMR spectra of PRP-1: ¹H (A), ¹³C (B), DEPT-135 (C), HSQC (D), COSY (E), HMBC (F), NOESY (G), and predicted structure (H).

Chemical shift assignment of PPRP-2

The monosaccharide composition and methylation results indicated the absence of fructose in PPRP-2, and the proportion of glucose was also significantly decreased, concomitant with an increase in Gal, GalA, Rha, and Ara. The ¹H NMR (Figure 5A) revealed anomeric signals in PPR-2 at δ 5.26, 5.25, 5.13, 5.10, 4.97, 4.65, and 4.44, suggesting the presence of both α and β glycosidic linkages [38]. The signal at δ 1.27 was a characteristic signal of Rha H-6. Furthermore, δ 3.82 represented the proton signal for -OMe, and the presence of δ 2.09 and 2.19 indicated that PPRP-2 may have acetyl substitution, with ¹³C NMR (Figure 5B) confirming possible methoxy and acetyl groups at δ 53.05 and 20.41, respectively. The signals at δ 176.72, 173.30, and 171.13 corresponded to the carbonyl signals of α-D-GalAp, the acetyl group, and methylated α-D-GalAp, respectively. Based on the ¹³C NMR (Figure 5B) and HSQC spectra, the anomeric signals included δ 5.24/109.31, 5.10/107.36, 5.14/107.12, 4.64/104.63, 4.44/103.69, 4.96/100.27, 5.10/99.29, and 5.26/98.37. By comparison with the methylation experiment results and the literature, these signals were assigned to α-L-Araf-(1→ (I), →5)-α-L-Araf-(1→ (G), →3, 5)-α-L-Araf-(1→ (K), →4)-β-D-Galp-(1→ (L), β-D-Galp-(1→ (M), →4)-α-D-GalAp-6-O-Me-(1→ (N), →4)-α-D-GalAp-(1→ (P), →2,4)-α-L-Rhap-(1→ (Q), and →4)-α-D-Glcp-(1→ (R) [39,40,41]. In the HMBC spectrum, correlations at δ 5.24/83.06 and 3.62/104.43 indicated potential linkages between H-1 of I and C-3 of K, and H-4 of R and C-1 of L. Additionally, δ 4.18/100.27, 4.39/77.79, 4.13/107.61, and 3.73/107.61 suggested possible linkages between H-6 of L and C-1 of N, H-4 of P and C-2 of Q, H-4 of Q and C-1 of J, and H-5 of K and C-1 of J. In the NOESY spectrum, signals at δ 5.10/4.46, 5.14/3.73, and 4.44/3.73 supported the presence of →4)-α-D-GalAp -(1 → 4)-α-D-GalAp-6-OMe-(1→, → 1)- α-L-Araf-(5 →1)- α-L-Araf-(3,5→, and 1)- α-L-Araf-(5 →1)- β-D-Galp. The likely structure of PPRP-2 is shown in Figure 5H. Similarly, the structure of PPRP-3 was elucidated (Figure S6).

Figure 5.

NMR spectra of PPRP-2: ¹H, (A) ¹³C, (B) DEPT-135 (C), HSQC, (D) COSY, (E) HMBC, (F) NOESY, (G), and predicted structure (H).

3.3. Physicochemical Property Characterization

3.3.1. Colorimetric Results

The color parameters (L*, a*, b*) serve as objective indicators of the degree of processing [9]. Table S2 showed the color changes in PRP and PPRP. In comparison with PRP, PPRP exhibited significantly lower lightness (L*) (p < 0.05), as well as higher a* (red–green) and b* (yellow–blue) values (p < 0.05). The ΔE* values (2.04–3.11) surpassed the reference threshold for perceptibility (0–2) [42], indicating visually perceptible color differences attributable to processing. These suggested that processing significantly affected the polysaccharide color, likely resulting from a Maillard reaction that generates natural pigments [43], with some free fructose potentially acting as reducing substrates.

3.3.2. Microstructural Characterization by SEM

SEM enables the direct visualization of morphological changes in polysaccharides induced by steaming. The SEM results for PRP and PPRP are illustrated in Figure 6. Both PRP-1 and PRP-2 appeared as dense, irregular blocks. PRP-1 possessed a slightly rough surface, whereas PRP-2 appeared smoother. The formation of this compact structure results from the close packing of polysaccharide molecular chains, a process facilitated by water sublimation during the freeze–drying process [44]. In contrast, PPRP-1 showed a smooth surface with irregular branching and spherical protrusions, while PPRP-2 and PPRP-3 displayed loose structures with rough, porous surfaces. The morphological differences are attributed to the structural alterations induced by high-temperature steaming. At higher magnifications, distinct bead-like structures were observed in PPRP-2 and PPRP-3. During this process, bound water rapidly vaporizes, generating steam pressure while the polysaccharide matrix softens; this enables the steam to expand the network into a porous structure [45,46]. Concurrent thermal effects degrade molecular chains, releasing volatiles that increase porosity, while cross-linking reactions stabilize the porous architecture [47].

Figure 6.

Scanning Electron Microscope (SEM) images of the polysaccharides at different magnifications (300×, 1000×, and 4000×).

The densely structured polysaccharides, such as PRP, may serve as ideal carriers for sustained or site-specific drug delivery due to their excellent biocompatibility and low toxicity [48,49]. Furthermore, their superior barrier properties make them promising candidates for food packaging applications [50]. In contrast, highly porous polysaccharides with large specific surface areas, exemplified by PPRP, can not only potentially enhance antioxidant activity but also improve the solubility of flavonoid and other active ingredients [51,52].

3.3.3. TG-DSC Analysis

Simultaneous TG-DSC, combining thermogravimetric (TG) and differential scanning calorimetric (DSC) techniques, provides critical insights into the thermal behavior, degradation pathways, and conformational dynamics of polysaccharides [53]. As the temperature increased, the polysaccharides exhibited three weight loss stages (Figure 7), and the detailed changes for each stage are shown in Table S3.

Figure 7.

TG-DSC analysis of polysaccharides: PRP-1 (A), PRP-2 (B), PPRP-1 (C), PPRP-2 (D), PPRP-3 (E).

In the initial stage, the weight loss of PRP-1, PRP-2, and PPRP-3 was approximately 7%, primarily due to the removal of physically adsorbed and partially bound water. In contrast, PPRP-1 and PPRP-2 exhibited weight losses of 11.39% and 21.82%, respectively. This indicated that, in addition to water loss, structural degradation occurred in both PPRP-1 and PPRP-2 prior to the main decomposition phase. Notably, for PPRP-2, the significant removal of unstable side chains led to a “purification” of the molecular structure, thereby enhancing a basis for enhanced thermal stability at elevated temperatures.

The subsequent main decomposition stage was crucial for evaluating the thermal stability of the materials [53]. Weight loss in this phase was attributed to the volatilization of CO, CO₂, and H₂O, resulting from the decomposition of saccharide rings and the cleavage of C-C and C-O bonds [54]. Based on a comprehensive evaluation of both onset temperature and mass loss, PPRP-2 displayed superior thermal stability: its onset temperature reached 257.70 °C, accompanied by the lowest weight loss at 39.69%, providing strong evidence of the effectiveness of its structural modifications. In contrast, the onset temperatures for PRP-1, PRP-2, and PPRP-1 ranged from 208 to 221 °C, with mass losses exceeding 58%. This suggested rapid decomposition upon the onset of degradation. PPRP-3 exhibited the lowest onset temperature (184.23 °C), indicating the poorest thermal stability; however, its mass loss (41.16%) was relatively low, suggesting a heterogeneous molecular structure in which one segment decomposes readily, while another is likely retained as a char-forming precursor.

The char formation stage ultimately determines the char yield of the material, involving intricate chemical rearrangements and potential graphitization of the residue [55]. PPRP-3 demonstrated markedly superior char-forming performance, resulting in a final yield of 31.79%, approximately an order of magnitude higher than that of other samples. In contrast, PPRP-2 exhibited a significant char yield (3.89%), while PRP-1, PRP-2, and PPRP-1 underwent nearly complete decomposition. Thus, PPRP-2 is classified as a “heat-resistant” material due to its stability during the primary decomposition stage, whereas PPRP-3 is identified as a distinct “high-char-yield” material. These properties render PPRP-3 suitable for the development of intelligent drug delivery systems, wound dressings, or food packaging where absorption, protection, and preservation are required.

3.4. Assessment of the Renal Protective Effects on NRK-52E Cells

3.4.1. Effects on NRK-52E Viability

As illustrated in Figure S7, cell viability was significantly reduced in the HG group compared with the NG group (76.82%, p < 0.05). In contrast to the HG group, the Met, PPRP-1, and PPRP-2 groups (12.5–100 μg/mL) significantly enhanced cell viability throughout the entire concentration range (p < 0.05). Conversely, PRP-1, PRP-2, and PPRP-3 exhibited a delayed concentration-dependent response, achieving significant increases in viability only at 25, 50, and 100 μg/mL (p < 0.05), respectively. At a concentration of 100 μg/mL, all treatments exhibited comparable effects (p > 0.05).

3.4.2. Oxidative Stress Assessment

HG treatment significantly increased oxidative stress in NRK-52E cells compared to the NG group, as evidenced by decreased levels of SOD and GSH and elevated MDA levels (p < 0.05). This oxidative damage was mitigated by polysaccharide treatment, with PPRP-2 (100 μg/mL) demonstrating significantly better efficacy than PRP-1 and PRP-2 in restoring SOD and GSH levels (Figure 8A–C).

Figure 8.

Effects of polysaccharides on SOD, GSH, MDA (A–C), and FN, COL1, α-SMA (D–F) in NRK-52E cells. (n = 3, experimental values are presented as mean ± SD, bars labeled with different letters indicate significantly different at p < 0.05). The x-axis represents the concentration of polysaccharides (μg/mL); control groups (NG, HG, and Met) are indicated.

3.4.3. Fibrogenic Protein Expression Analysis

FN, COL1, and α-SMA are pivotal biomarkers of renal fibrosis, indicating extracellular matrix disruption, scar formation, and fibroblast activation, respectively [56,57]. Under HG conditions, NRK-52E cells undergo epithelial–mesenchymal transition, leading to abnormal expression of these proteins [25]. Consequently, the levels of FN, COL1, and α-SMA serve as critical indicators for evaluating the anti-fibrotic potential of the polysaccharides. Compared to the NG group, the HG group demonstrated a significant upregulation of FN, COL1, and α-SMA (Figure 8D–F). The polysaccharides significantly alleviated HG-induced fibrotic damage in NRK-52E cells, with PPRP-2 (100 μg/mL) exhibiting superior efficacy over PRP-1 and PRP-2 in reducing COL1 expression (p < 0.05).

In summary, the enhanced nephroprotective effect of PPRP-2 in HG-induced NRK-52E cells likely arises from its complex monosaccharide composition, moderate uronic acid content, and molecular weight, all of which augment its antioxidant capacity [11,58,59,60]. Moreover, the reduced degree of esterification in PPRP-2 (0.76) relative to PPRP-3 (0.92) resulted in an increased negative charge density, thereby enhancing antioxidant activity [61]. Thus, the suppression of renal fibrosis can be achieved by alleviating oxidative stress, which in turn delays the progression of various chronic kidney diseases, including DKD [62]. Although PPRP-3 shared a similar monosaccharide composition with PPRP-2, its relatively higher Mw (21.5 kDa) significantly hindered cellular uptake [63].

Therefore, this study not only revealed the material basis for the enhanced activity of PPRP but also highlighted its significant application potential as a medicinal food homology material. Given their demonstrated renoprotective activity, they can be developed into daily dietary supplements or functional food ingredients targeting kidney health, particularly for populations at risk of or managing DKD. This provides a solid foundation for the use of medicinal food homology materials in specific chronic disease support. From an industrial perspective, the steam processing method employed in this study offers inherent advantages in safety and sustainability, avoiding chemical residues and pollution issues associated with conventional chemical modifications. This presents an attractive technical pathway for the development of high-value-added natural health products.

4. Conclusions

Through a systematic comparison of polysaccharides derived from raw and processed P. sibiricum (PRP and PPRP), this study elucidated the structural basis for the enhanced bioactivity attributed to traditional steaming. The primary structures of PRP-1 and PRP-2 were identified as fructans with backbones consisting →1)-β-D-Fruf-(2→ linkages. Notably, steam processing altered the structures of polysaccharides, resulting in simultaneous degradation and aggregation of their molecules. PPRP-1 was selectively modified with additional →5)-α-L-Araf-(1→, while PPRP-2 and PPRP-3 underwent fundamental restructuring, transitioning from a fructan to a pectin-like backbone characterized by →4)-α-D-GalAp-(1→ (in both methylated and non-methylated forms), →4)-β-D-Galp-(1→, →5)-α-L-Araf-(1→, and →2,4)-α-L-Rhap-(1→., among others. Physicochemical characterization revealed that processing induced color darkening and transformed the polysaccharides into porous structures, with PPRP-2 exhibiting enhanced thermal stability compared to PRP. All five purified polysaccharides significantly protected NRK-52E cells from HG-induced injury by enhancing viability and attenuating renal damage, with PPRP-2 exhibiting superior efficacy. These variations in bioactivity were strongly correlated with distinct structural parameters, particularly monosaccharide composition and molecular weight. These findings provide a theoretical basis for explaining the efficacy enhancement of PPRP and promoting its application in functional foods.

Abbreviations

The following abbreviations are used in this manuscript:

Ara	Arabinose
BSA	Bovine serum albumin
COL1A	Collagen type I
DKD	Diabetic kidney disease
DMEM	Dulbecco’s modified Eagle’s medium
DSC	Differential scanning calorimetric
DTNB	5,5′-dithiobis (2-nitrobenzoic acid)
ESRD	End-stage renal disease
FBS	Fetal bovine serum
FN	Fibronectin
Fru	Fructose
FT-IR	Fourier-transform infrared spectroscopy
Gal	Galactose
GalA	Galacturonic acid
Glc	Glucose
HG	High glucose
HPGPC	High performance gel permeation chromatography
IC	Ion chromatography
Met	Metformin HCl
Mw	Molecular weight
NG	Normal glucose
PMAA	Partially methylated alditol acetate
PPR	Processed Polygonatum Rhizome
PPRP	Polysaccharides of processed Polygonatum Rhizome
PR	The rhizome of Polygonatum Rhizome
PRP	The polysaccharides of Polygonatum Rhizome
Rha	Rhamnose
RAS	Renin-angiotensin system inhibitors
SEM	Scanning electron microscope
SGLT2	Sodium-glucose cotransporter-2
TBA	Thiobarbituric acid
TCM	Traditional Chinese Medicine
TFA	Trifluoroacetic acid
TG	Thermogravimetric
TG-DSC	Thermogravimetric differential scanning calorimetry analyzer
WST-1	Water-soluble tetrazolium salt
α-SMA	A-smooth muscle actin

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/foods15040619/s1, Figure S1: The ultraviolet spectra of polysaccharides; Figure S2: Molecular weight distribution of polysaccharides determined by HPGPC: PRP-1 (A), PRP-2 (B), PPRP-1 (C), PPRP-2 (D), PPRP-3 (E); Figure S3: Congo red experimental analysis of polysaccharides; Figure S4: NMR spectra of PRP-2: ¹H (A), ¹³C (B), DEPT-135 (C), HSQC (D), COSY (E), HMBC (F), NOESY (G), predicted structure (H); Figure S5: NMR spectra of PPRP-1: ¹H (A), ¹³C (B), DEPT-135 (C), HSQC (D), COSY (E), HMBC (F), NOESY (G), predicted structure (H); Figure S6: NMR spectra of PPRP-3: ¹H (A), ¹³C (B), DEPT-135 (C), HSQC (D), COSY (E), HMBC (F), NOESY (G), predicted structure (H); Figure S7: Effects of polysaccharides on cell viability; Table S1. ¹H and ¹³C NMR chemical shifts in the polysaccharides. Table S2: The color values of polysaccharides; Table S3: Heat loss process of polysaccharides.

Author Contributions

Conceptualization, X.L.; methodology, H.J. and S.H.; software, H.J. and A.W.; formal analysis, H.J.; investigation, H.J. and S.H.; resources, X.L.; data curation, H.J.; writing—original draft preparation, H.J.; writing—review and editing, D.W., Y.P., S.R., K.R. and X.L.; visualization, H.J., A.W. and Z.L.; supervision, X.L.; project administration, K.R.; funding acquisition, X.L., Y.P. and K.R. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Materials. Further inquiries can be directed to the corresponding authors.

Conflicts of Interest

The authors declare no conflicts of interest.

Funding Statement

This work was supported by the National Natural Science Foundation of China (81973465), the 2023 Basic Research Project of Colleges and Universities of Liaoning Provincial Department of Education (JYTZD2023137), the Liaoning Province Science and Technology Plan Joint Plan (2024-MSLH-448) and the Faculty Resource Research Initiation Programs for Newly Introduced PhDs of Shenyang Pharmaceutical University (2411520022).