Physicochemical characterization and immune activity of water-extract polysaccharides by stepwise ethanol precipitation from wild Cordyceps sinensis

Abstract

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Cordyceps sinensis is widely utilized in China as an edible and medicinal fungus for the treatment of immunodeficiency-related disorders. Evidence suggests that polysaccharides are the principal bioactive components responsible for its immunostimulatory effects. However, the physicochemical properties, bioactivities, and structure–function relationships of these polysaccharides remain inadequately elucidated. In this study, four distinct crude polysaccharides from wild Cordyceps sinensis (WCP) were isolated using stepwise ethanol precipitation at final concentrations of 20%, 40%, 60%, and 80% (v/v). These fractions were designated as WCP-20, WCP-40, WCP-60, and WCP-80, respectively. Results demonstrated that the molecular weight (MW) of WCP decreased as the ethanol concentration increased. High-MW fractions (WCP-20/-40) exhibited high glucose content, a partially triple-helical structure, levorotation (−), and greater thermal stability. In contrast, the low-MW fractions (WCP-60/-80) were enriched in galactose and mannose, exhibited a higher branching density, and dextrorotation ( +). Furthermore, this study revealed that WCPs activated macrophages by enhancing phagocytosis and stimulating the secretion of nitric oxide and interleukin-1 beta. These immunostimulatory effects were mediated through the MAPK/NF-κB signaling pathway. Specifically, WCP-20 triggered macrophage activation via the ERK/JNK/p65 pathway, with P38/ERK/JNK pathway for WCP-40, P38/ERK/JNK/p65 pathway for WCP-60, and JNK/p65 pathway for WCP-80. Correlation analysis revealed that the immunostimulatory effects of WCPs were closely linked to their monosaccharide composition and secondary structures. These findings established that the physicochemical properties of WCP were critical determinants of precise immune modulation. This study provided a foundational reference for developing precision polysaccharide-based immune-enhancing nutraceuticals.

Graphical Abstract

Introduction

Cordyceps sinensis (synonymous with Ophiocordyceps sinensis) is a recognized medicinal material listed in the Chinese Pharmacopoeia [1]. It consists of a symbiotic complex formed by the fungal stroma and the deceased caterpillar. According to international mycological nomenclature, the fungus is properly identified as Ophiocordyceps sinensis (Berk.) Sung, Sung, Hywel-Jones, and Spatafora [2]. Extensive pharmacological studies have demonstrated that Cordyceps sinensis (C. sinensis) possesses a wide range of biological activities, among which immunomodulation is one of the most thoroughly investigated and therapeutically promising [3]. Research has also identified polysaccharides as the primary bioactive constituents responsible for the immunomodulatory properties of C. sinensis [4, 5]. Cordyceps polysaccharides exhibit potent abilities to modulate immune cell functions, particularly macrophage activation and cytokine regulation, highlighting their significant potential for use in ethnomedicinal immunotherapies [6].

As central effector cells of innate immunity, macrophages represent a major target for Cordyceps polysaccharides [7]. Their activation, polarization, and cytokine production are intricately regulated by key signaling pathways, notably the mitogen-activated protein kinase (MAPK) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathways [8]. Previous research has isolated a glucan (CCP, MW = 433.8 kDa) from C. sinensis that stimulates macrophage secretion of IL-6 and TNF-α through activation of the TLR4/MyD88/P38 signaling pathway [9]. Another polysaccharide (CME-1) obtained from fermented C. sinensis mycelia suppresses B16F10 melanoma cell migration by suppressing ERK/P38 phosphorylation and upregulating IκBα expression [10]. It also attenuates lipopolysaccharide (LPS)-induced expression of inducible nitric oxide synthase (iNOS) in macrophages via ceramide-mediated activation of protein phosphatase 2A [11]. These findings collectively illustrate the capacity of Cordyceps polysaccharides to activate these pathways, influencing both pro-inflammatory and anti-inflammatory responses.

Graded ethanol precipitation is a widely adopted technique for separating and purifying polysaccharide fractions, offering both economic and environmental advantages [12]. This method enables efficient fractionation of crude polysaccharides into distinct molecular weight (MW) components [12]. When integrated with biotechnology, this method allows for the identification of polysaccharide fractions with optimal pharmacological profiles, supporting the development of therapies for anti-tumor [13], dermoprotective [14], hypolipidemic [15], and other applications. This approach also promotes the development of high-value agricultural by-products. In China’s Tibetan regions, C. sinensis is traded as a commercial agricultural product and represents a crucial source of income for pastoral communities across the Qinghai-Tibet Plateau, Nepal, and Bhutan [16, 17]. Despite its potential, the combined use of graded ethanol precipitation and bioactivity evaluation has not been extensively applied to the study of immunomodulatory polysaccharides from natural O. sinensis polysaccharides.

As complex biomacromolecules, the bioactivity of Cordyceps polysaccharides is inherently connected to their intricate structural characteristics. However, most structural and mechanistic investigations have focused on polysaccharides derived from fermented mycelia rather than those from wild C. sinensis [18, 19]. Cordyceps polysaccharides derived from C. sinensis exhibit complex compositions and generally have a higher MW. Current understanding of their physicochemical properties remains limited, with comparative studies across different MW fractions being particularly scarce. Therefore, this study aimed to isolate and characterize wild Cordyceps polysaccharides (WCP) with distinct MW distributions, elucidate the immunomodulatory effects on macrophages, and the underlying mechanisms involving the MAPK/NF-κB signaling pathway using targeted pathway inhibitors. Another key objective was to conduct comprehensive correlation analyses to establish correlation by linking the structural characteristics of WCP to its  immunoregulatory potency and specific pathway activation profiles. The findings are expected to provide a scientific basis for the development of ethnopharmaceuticals and immune-targeted dietary supplements derived from wild C. sinensis.

Materials and methods

Chemicals and reagents

Wild C. sinensis specimens were collected and purchased from local farmers in Yajiageng Village, Kangding City, Sichuan Province (longitude: 101.97°-102.01° E, latitude: 29.88°-29.92° N; altitude: 3400–4100 m). A voucher specimen (Voucher Number: 20230502001) has been deposited at the Herbarium of Chengdu University of TCM (Fig. S1). RAW264.7 cells were acquired from the Shanghai Cell Bank of the Chinese Academy of Sciences. The following reagents were used in this study: CCK-8 kit (US Everbright, #006Z0102); Neutral Red (Beyotime, #P0011); RNA Extraction Kit (Vazyme, #R701-01); Fast SYBR Green qPCR Master Mix UDG (#A402-01) and ExonScript RT SuperMix (#A502-01) from Chengdu Rongwei Gene; Annexin V-FITC Apoptosis Kit (Beyotime, #021921210630); LPS (Gibco™, #L4515). Antibodies used in this study include ERK (ZEN-BIO, #343830), p-ERK (Affinity, #AF1015), P38 (ABclonal, #A0227), p-P38 (ABclonal, #AP1502), JNK (Proteintech, #AF6318), p-JNK (Proteintech, #AF3318), p65 (Proteintech, #80979-1-RR), and p-p65 (Affinity, #AF2006). Inhibitors included SB203580 (Abmole, #M1781-202405), SP600125 (Abmole, #M2076-202406), PD98059 (MCE, #HY-12028), and BAY11-7082 (Abmole, #M2040-202404).

Polysaccharide extraction and fractional precipitation protocol

Wild C. sinensis was dried and pulverized into a fine powder using a cryogenic grinder. The powder was subjected to three rounds of hot water extraction at 80 °C (1:30 w/v) for 2 h per round . The supernatants were pooled following centrifugation at 5,000 × g for 10 min to remove insoluble debris. The combined extract was concentrated under reduced pressure at 60 °C. Polysaccharides were precipitated by adding four volumes of absolute ethanol and allowing the mixture to stand overnight at 4 °C. The precipitate was redissolved in pure water and deproteinized using the Sevag method (chloroform: butanol = 4: 1). This deproteinization was repeated until no protein impurities were detectable. The solution was dialyzed against deionized water for 72 h using a membrane with a 3.5 kDa MW cutoff. The total polysaccharides were finally obtained via freeze-drying at - 50 °C.

A solution of total polysaccharides was prepared at 40 mg/mL and subsequently mixed with absolute ethanol to a final concentration of 20% (v/v). After storage at 4 °C for 12 h, the mixture was centrifuged. The pellet was re-dissolved in water and then lyophilized to obtain WCP-20. The supernatant was subsequently treated with anhydrous ethanol to achieve a 40% (v/v) ethanol concentration. Using the same sedimentation and isolation procedure, WCP-40 was collected. Further sequential precipitation at 60% and 80% (v/v) ethanol concentrations yielded WCP-60 and WCP-80, respectively (Fig. 1). The recovery yield for each fraction was calculated as the percentage of its freeze-dried mass relative to the total mass of the initial crude polysaccharides.

Fig. 1

Extraction and ethanol-grade precipitation for polysaccharide components from wild C. sinensis

Characteristic analysis of WCP

Gel permeation chromatography (GPC) and dynamic light scattering (DLS) analysis

Approximately 0.20 mg of each sample was dissolved in 1 mL of ultrapure water, thoroughly mixed, and filtered through a 0.22 μm membrane. A 100μL aliquot of the filtrate was drawn using a microsyringe and injected into the GPC system. Particle size analysis of the polysaccharide solution (1.0 mg/mL) was performed using DLS system.

Determination of total sugar, protein, and uronic acid

The total sugar content in the sample was quantified using the phenol–sulfuric acid method, with glucose serving as the standard for calibration [20]. The uronic acid content in the polysaccharide fractions was determined via the carbazole–sulfuric acid colorimetric method, with a glucuronic acid standard solution utilized to plot the standard curve [21]. Protein content was measured with the bicinchoninic acid (BCA) assay, calibrated against bovine serum albumin [22].

Ultraviolet–visible spectroscopy (UV–Vis) scanning

Sample solutions (1.0 mg/mL) were prepared in ultrapure water and analyzed in quartz cuvettes with a UV–Vis spectrophotometer, using ultrapure water as the blank. Absorbance was scanned across a wavelength range of 200–800 nm.

Determination of monosaccharide composition

Monosaccharide composition was determined as described previously  [23]. Briefly, 5 mg of each sample was hydrolyzed with 1 mL of 2.5 M trifluoroacetic acid (TFA) at 121 °C for 2 h. Excess TFA was removed by repeated co-evaporation with methanol under a nitrogen stream. The dried hydrolysate was reconstituted in distilled water and analyzed by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) using an ICS-5000 system (Thermo Fisher Scientific) equipped with a Dionex CarboPac PA20 column (150 mm × 3 mm, 6.5 μm).

Fourier transform-infrared (FT-IR) spectrometry

For FTIR analysis, 1 mg of each WCP sample was homogenized with 150 mg of KBr and pressed into a transparent pellet. Spectra were acquired using a Nicolet iS10 spectrophotometer (Waltham, MA, USA) over the spectral range of 4000 to 400 cm^⁻1.

Specific rotation test

A 2.5-mL aliquot of each WCP solution (0.2 mg/mL) was transferred into a polarimetric cell with a 10-cm path length. Specific optical rotation was measured at 20 °C and a wavelength of 589 nm.

Scanning electron microscopy (SEM) analysis

Dried polysaccharide samples (about 2.0 mg) were mounted on conductive adhesive tape and sputter-coated with gold under vacuum. Surface morphology was examined using SEM at various magnifications.

Circular dichroism (CD) spectrometer analysis

WCP samples were dissolved in ultrapure water to a concentration of 0.5 mg/mL. A 0.4 mL aliquot was transferred to a quartz cuvette and scanned from 200 to 400 nm using a CD spectrometer. Secondary structure elements (e.g., helices, β-sheets, β-turns, random coils) were quantified with CDNN software.

Congo red experiment

Samples were prepared as solutions with a concentration of 1.0 mg/mL, while the concentration of the Congo red solution was set at 80 μ mol/L. Sodium hydroxide was prepared in varying concentrations of 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 mol/L. The polysaccharide solution, Congo red solution, and sodium hydroxide solution were mixed in a 1:1:2 volume ratio and thoroughly agitated. After incubation at room temperature for 10 min, the absorption spectra were recorded between 400 and 600 nm. A curve of λ_max versus NaOH concentration was plotted [24].

Iodine–potassium iodide test

Samples (10 mg) were dissolved in ultrapure water to prepare a solution with a concentration of 1 mg/mL. After adding 1.2 mL of iodine reagent (0.02% I₂, 0.2% KI) to 2 mL of the sample, the mixture was vortexed and subsequently scanned from 200 to 700 nm using the  UV-Vis spectrophotometer .

Thermogravimetric analysis

Samples (5.0 mg) were placed into crucibles and analyzed using thermogravimetry at a nitrogen flow rate of 25 mL/min. The temperature increases from 20 °C to 800 °C at a rate of 10 °C per minute.

Effect of WCP on RAW 264.7 cells

Effect of WCP on the viability of RAW 264.7 cells

RAW264.7 cells were cultured in complete medium (10% FBS, 1% penicillin–streptomycin) and seeded into 96-well plates at 1 × 10⁵ cells per well and cultured until they reached 90% confluence. A negative control (NC) group and groups treated with WCP were established. The WCP-treated groups were incubated with WCP-20, WCP-40, WCP-60, or WCP-80 at concentrations of 2, 20, 100, and 200 µg/mL for 24 h. The negative control (NC) received phosphate buffer saline (PBS) alone. Cells were then incubated with 10% CCK-8 solution for 30 min at 37 °C in 5% CO₂. Absorbance was measured at 450 nm. Cell viability was calculated as: Cell proliferation rate = [(OD _experimental − OD _blank)/(OD _control − OD _blank)] × 100%. OD _blank represents the combined background absorbance of the CCK-8 solution and the 96-well plate.

Effect of WCP on the immune function of RAW 264.7 cells

Experimental group design

RAW264.7 cells were divided into: a negative control (NC), LPS-positive control (1 μg/mL), and WCP-treated groups (WCP-20, WCP-40, WCP-60, WCP-80), corresponding to low, medium, and high doses (L/M/H: 2, 20, and 100 μg/mL, respectively).

Cell phagocytic ability test

Logarithmic-phase cells were seeded at a denssectity of 1 × 10⁵ cells per well in 96-well plates. The cells were treated for 24 h according to the protocol described in Sect. 2.4.2.1, after which the media was replaced with 200 μL of fresh media containing 20 μL of neutral red saline solution (0.1% w/v) and incubated for an additional 2 h. The supernatants were then discarded, and the cells were washed three times with PBS. Subsequently, 200 μL of cell lysis buffer (ethanol: glacial acetic acid = 1:1) was added to each well. After 10 min, the absorbance at 540 nm (OD₅₄₀) was measured using a microplate reader. The phagocytosis rate was calculated using the following formula: phagocytosis rate = [(OD _experimental – OD _blank) / (OD _control – OD _blank)] × 100%, where OD _blank represents the combined background absorbance of the cell lysis buffer and the 96-well plate.

For the Jurkat cell phagocytosis assay: RAW264.7 cells (1 × 10⁵/well) were prepared as described in Sect. 2.4.2.1, and co-cultured with apoptotic Jurkat cells (pre-stained with Cell-Tracker Green and UV-irradiated) at a 5: 1 ratio for 2 h [25] (Fig. S2). Non-phagocytosed cells were removed by washing with PBS. Cells were then stained with F4/80 at 4 °C in the dark for 30 min and analyzed by flow cytometry.

Detection of nitric oxide (NO) secretion by the Griess method

Logarithmically growing cells were seeded at a density of 5 × 10⁵ cells per well in a 24-well plate. After 24 h of treatment, the supernatant was collected. Then, 50 μL of supernatant was transferred to each well of a 96-well plate, mixed thoroughly with 50 μL of Griess reagent I, followed by the addition of 50 μL of Griess reagent II. After thorough mixing, absorbance was measured at 540 nm using a microplate reader.

Detection of interleukin-1 β (IL-1β) secretion by ELISA

Logarithmically growing cells were seeded at a density of 1 × 10⁵ cells per well in a 96-well cell culture plate. Following 24 h of culture with the corresponding drugs, the supernatant was collected, and IL-1β concentration was measured according to the ELISA kit instructions.

Detection of Nos2 and Il1b expression by RT-qPCR

Total RNA was extracted after 24 h of treatment using the RNeasy separation reagent. The extracted RNA was subsequently dissolved. Genomic DNA removal and reverse transcription were conducted using a dsDNase-containing kit. One microgram of RNA was incubated with oligo (dT) primers at 25 °C for 10 min, cDNA synthesis was then performed  at 55 °C for 15 min. Gene-specific primers were designed based on the NCBI database, and their sequences are listed in Table S1. qPCR amplification was performed using SYBR Green Master Mix (20 μL system: 10 μL premix, 0.4 μM primers each, 1 μL cDNA template). The amplification conditions included an initial denaturation at 95 °C for 10 min, followed by 40 cycles of 95 °C/5 s and 60 °C/30 s, with melting curve analysis for specificity verification. Relative gene expression was normalized to GAPDH and calculated using the 2^−ΔΔCt method.

Detection of MAPK/NF-κB pathway protein expression by Western blot

After 24 h of drug treatment, 500 μL of pre-cooled RIPA lysis buffer (containing 1% protease inhibitor and 1% phosphatase inhibitor) was added, and the cells were homogenized at 4 °C. The mixture was then placed on ice for 30 min. The samples were then centrifuged at 12,000 × g for 10 min. Protein concentrations were quantified using the BCA assay and were subsequently adjusted to 2 mg/mL. Samples were then mixed with 5 × loading buffer and denatured by boiling for 5 min. Electrophoresis was conducted at 80 V for 20 min, followed by 120 V for 90 min. Proteins were subsequently transferred onto membranes at a constant current of 200 mA for 65 min. The membranes were then probed with the following primary antibodies (all used at 1: 1000 dilution): P38, p-P38, ERK, p-ERK, JNK, p-JNK, p65, p-p65, and β-actin. Subsequently, the membranes were incubated with an HRP-conjugated  secondary antibody (1:5000). Detection was conducted using the ECL chemiluminescence system. Target protein levels relative to the internal control were analyzed using ImageJ software.

Statistical analysis

Data were analyzed using GraphPad Prism and are presented as the mean ± standard deviation (SD). For comparisons among multiple groups, a one-way ANOVA followed by the least significant difference (LSD) post hoc test was used when the variances were equal; otherwise, the Kruskal–Wallis H test was applied. Differences were considered statistically significant at P < 0.05. Heatmaps were generated using https://www.bioinformatics.com.cn, while correlation analysis was performed using OmicStudio (https://www.omicstudio.cn/tool).

Results

Extraction of the polysaccharides from wild C. sinensis

Total polysaccharides were isolated from wild C. sinensis by a single round of hot-water extraction, yielding approximately 2.39% (w/w) relative to the dry starting material. Following deproteinization using the Sevag method and subsequent dialysis to remove low-MW compounds, the dialyzed crude polysaccharide solution was fractionated through graded ethanol precipitation at concentrations of 20%, 40%, 60%, and 80% (v/v). This process yielded four fractions, designated as WCP-20, WCP-40, WCP-60, and WCP-80. Polysaccharide recovery rates from the dialyzed solution were 23.38%, 7.59%, 17.40%, and 46.10%, respectively. Correspondingly, the overall yields of each fraction, based on the dry weight of the C. sinensis powder, were approximately 0.56%, 0.18%, 0.42%, and 1.10%, respectively (Table 1).

Table 1 Yield and physicochemical properties of WCPs

Identification and composition analysis

The total sugar content of the four crude Cordyceps polysaccharide fractions, determined by the phenol–sulfuric acid method with glucose as the standard, decreased in the following order: WCP-80 > WCP-20 > WCP-60 > WCP-40. In all fractions, the UV-Vis spectra displayed faint absorption peaks at 280 nm, indicating trace amounts of protein. BCA assay quantification confirmed low protein levels of approximately 5% across the samples (Table 1; Fig. 2A). In contrast, negligible absorption was observed at 260 nm, confirming the absence of nucleic acids.

Fig. 2

The physicochemical properties of WCP. A UV spectra analysis. B Molecular weight distribution curve. C Particle size analysis. D Monosaccharide composition analysis. Monosaccharide peak assignments: 1, fucose; 2, rhamnose; 3, arabinose; 4, galactose; 5, glucose; 6, xylose; 7, mannose; 8, fructose; 9, ribose; 10, galacturonic acid; 11, guluronic acid; 12, glucuronic acid; 13, mannuronic acid. E FT-IR analysis. F Iodine–potassium iodide test. G Circular dichroism analysis. Color-based abundance comparison in clustering requires shared secondary structure. H Congo red test. I XRD analysis

GPC analysis revealed symmetrical, monomodal peaks for all  WCP fraction except WCP-20, indicating relatively uniform MW distributions (Fig. 2B). MW progressively decreased with higher ethanol concentrations during precipitation: WCP-20 > WCP-40 > WCP-60 > WCP-80 (Table 1). This inverse relationship between MW and the ethanol concentration aligns with established principles of polysaccharide fractionation [15, 26]. The polydispersity index (Mw/Mn) was higher in WCP-20 and WCP-40, indicating broader MW distributions within these fractions, whereas WCP-60 and WCP-80 showed lower Mw/Mn values close to 1.5, reflecting greater homogeneity.

DLS analysis was conducted to assess the solution behavior of the WCPs. As shown in Fig. 2C, the observation of Tyndall effects confirmed the colloidal nature of the solution. DLS measurement showed a positive correlation between the MW and the hydrodynamic diameter (Dh), with higher-MW fractions exhibiting larger Dh values (Table 1). Furthermore, the polydispersity index obtained from DLS intensity distributions also rose with increasing MW, suggesting broader particle size distributions in higher-MW fractions. These findings indicate that higher-MW WCPs adopt larger conformations or assemble into more heterogeneous aggregates in solution compared to lower-MW fractions.

Monosaccharide composition analysis revealed that all WCP fractions primarily consisted of glucose (Glc), mannose (Man), and galactose (Gal), consistent with previous reports on wild  Cordyceps polysaccharides [7]. Small amounts of glucuronic acid (GlcA) were also detected in all fractions (Fig. 2D). Interestingly, glucose content decreased significantly as MW decreased (WCP-20 > WCP-40 > WCP-60 > WCP-80). In contrast, mannose and galactose contents increased with decreasing MW (WCP-20 < WCP-40 < WCP-60 < WCP-80). Thus, higher-MW WCPs contained higher glucose levels but lower proportions of mannose and galactose.

FT-IR spectroscopy and specific optical rotation analysis

The FT-IR spectra of the four WCP fractions exhibited typical carbohydrate-associated absorption bands (Fig. 2E). All samples showed absorption peaks at around 3400 cm⁻¹ (O–H bonds) and 2930 cm⁻¹ (C-H bonds), along with a peak at 1040 cm⁻¹, the latter corresponding to the vibrations of C–O–H/C–O–C glycosidic bonds [15]. The prominent absorption peak at 1053 cm⁻¹ is attributed to the stretching vibration of the pyranose ring [27]. Notably, the faint signal at 972 cm⁻¹ observed in WCP-40, WCP-60, and WCP-80 may correspond to the C–O–C vibrations of pyranoside linkages, whereas no such signal appeared in WCP-20. Weak signals at 888 cm⁻¹ and 811 cm⁻¹ in WCP-20 and WCP-60 correspond to the β- and α-conformations, respectively. In contrast, WCP-40 and WCP-80 exhibit only a weak signal at 811 cm⁻¹, indicating the exclusive presence of α-conformations [28, 29]. Additional peaks at 1655, 1540, and 1314 cm⁻¹ corresponded to amide I, II, and III, suggesting trace proteins [30].

Optical rotation analysis reflected the influence of monosaccharide asymmetry and macromolecular chirality [31]. WCP-20 and WCP-40 exhibited dextrorotation in pure water, while WCP-60 and WCP-80 displayed levorotation (Table 1). The absolute values of optical rotation increased in the following order: WCP-20 < WCP-40 < WCP-60 < WCP-80, implying greater structural asymmetry in lower-MW polysaccharides.

Conformational analysis and molecular interaction study

Iodine–potassium iodide test

Polysaccharides can form complexes with iodine, which exhibit absorption peaks between 300 and 500 nm, reflecting branched or long side-chain structures. Absorbance at 565 nm indicates a reduced number of branches [32]. As shown in Fig. 2F, WCP-20, WCP-40, and WCP-60 exhibited absorbance peaks in the 300 to 350 nm range, with no detectable peak at 565 nm. This observation confirms the presence of long side chains and significant branching. In contrast, WCP-80 displayed the weakest absorption peak, suggesting minimal branching.

CD spectroscopy analysis

CD spectroscopy—a technique measuring the differential absorption of circularly polarized light by chiral molecules—,was used to probe secondary structures in the WCP. The spectral profiles (Fig. 2G) revealed distinct conformational differences. WCP-20 and WCP-40 predominantly adopted random coil conformations with substantial β-turn contributions. WCP-60 primarily exhibited a helical structure, though random coils were also present. WCP-80 displayed a mixed profile of random coils and helices. Cluster analysis with heat mapping further highlighted structural similarities between WCP-20 and WCP-40, as well as between WCP-60 and WCP-80.

Congo red test

The Congo red assay was used to assess triple-helical structures [33]. As shown in Fig. 2H, at NaOH concentrations of ≤ 0.1 mol/L, Congo red alone exhibited a blue shift. With NaOH concentrations increasing from 0.1 to 0.3 mol/L, the WCP–Congo red complexes showed concentration-dependent blue shifts. The extent of this shift decreased in the order: WCP-20 > WCP-40 > WCP-60 ≈ WCP-80 ≈ Congo red. Consequently, it can be inferred that WCP-20 and WCP-40 likely possess a triple-helical structure, suggesting that high-MW polysaccharides are more prone to forming such conformational arrangements.

Physical structure and microscopic morphology observation

XRD analysis

XRD analysis was conducted to assess the crystallinity of the WCP fractions. In general, crystalline domains yield sharp and narrow diffraction peaks, whereas amorphous structures produce broad and diffuse halos [34]. As shown in Fig. 2I, all WCP samples exhibited a broad, amorphous scattering halo in the range of 14.9° to 31.6°, accompanied by minor sharp peaks at 42.8°, 43.7°, and 49.6°. Among the fractions, WCP-40 showed an additional sharp crystalline peak at 29.46° and a broad band ranging from 36.6° to 45.8°. While WCP-60 and WCP-80 displayed a distinct sharp peak at 19.02°. All samples were primarily amorphous and exhibited low crystallinity. The relative diffraction intensities followed the order: WCP-20 > WCP-60 > WCP-40 > WCP-80 (Table 1).

Morphological observation

SEM provided detailed observations of the microstructures. All WCP samples displayed lamellar, sheet-like, or fibrous aggregates interspersed with pebble-shaped or rod-like particles (Fig. 3A). The four polysaccharides exhibited voids characterized by heterogeneous density and varying pore sizes. WCP-20 and WCP-40 predominantly exhibited tightly interconnected sheets, while WCP-60 and WCP-80 contained smaller and more dispersed sheets. At 100 × magnification, circular voids were visible on the surfaces. WCP-20 contained larger and sparsely distributed voids (20–50 μm), whereas WCP-40, WCP-60, and WCP-80 exhibited smaller (2–4 μm) and denser voids. At 1000× magnification, pores in WCP-20 became indistinct but remained evident in the other samples. At 2000 × magnification, WCP-20 and WCP-40 surfaces appeared smoother, while WCP-60 and WCP-80 exhibited wrinkled or frosted textures. Higher-MW fractions formed larger lamellar assemblies, whereas lower-MW samples tended toward more fibrous morphologies.

Fig. 3

Morphological observation and thermostability of the WCP. A SEM micrographs. B TG curve. C DTG curve

Thermostability analysis

Thermal stability was investigated by thermogravimetric analysis (Fig. 3B, C). The initial stage (25.0–255.6 °C) demonstrated a slight weight loss of approximately 10%, mainly due to water evaporation or the release of volatile molecules, indicating stability under ambient conditions. The second stage (255.6–345.8 °C) involved rapid decomposition, with 40.7–50.4% mass loss attributed to the cleavage of the carbon backbone and hydrogen bonds. WCP-60 decomposed first at 289.7 °C, followed by WCP-80 (peak at 296.5 °C and the highest decomposition rate). WCP-20 (peak at 307.2 °C), and WCP-40 (peak at 317.7 °C, highest thermal stability). The final stage (above 350 °C) involves the slow degradation of residual char, resulting in a 14–21% loss and the formation of thermally stable inorganics. Overall, higher-MW WCP fractions exhibited greater thermal resistance.

WCP enhances the immune activity of RAW 264.7 cells

Before evaluating the immunoactivating effects of WCP on RAW 264.7 cells, this study assessed the cytotoxicity of WCP toward RAW 264.7 cell  using the CCK-8 assay. As shown in Fig. 4A, WCP-20, WCP-40, and WCP-80 exhibited no cytotoxicity at concentrations from 2 to 200 μg/mL. However, WCP-60 reduced viability only at 200 μg/mL (P < 0.05). Based on these observations, 2, 20, and 100 μg/mL were chosen as low (L), medium (M), and high (H) doses for subsequent assays.

Fig. 4

Immunomodulatory effects of WCP on RAW 264.7 cells. A Macrophage proliferation. B Neutral red uptake by macrophages. C, D Phagocytosis of apoptotic Jurkat cells. E, F Gene expression levels of Nos2 and Il1b. E, F Secretion of NO and IL-1β. Results were expressed as mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 compared to the NC group

To assess the immunomodulatory effects of the four WCP fractions on macrophage function, this study quantified phagocytic activity using the Neutral Red assay (Fig. 4B). Comparable with LPS stimulation, all WCP fractions significantly enhanced macrophage phagocytosis in a dose-dependent manner (P < 0.05). At a concentration of 100 μg/mL, WCP-20, WCP-40, WCP-60, and WCP-80 increased phagocytosis to 186.7%, 163.5%, 170.7%, and 166.2% of control levels, respectively (P < 0.0001). Experiments involving co-culture of RAW264.7 cells with apoptotic Jurkat cells showed that only high-dose WCP-20 and mid-dose WCP-40 enhanced efferocytosis (P < 0.05, Fig. 4C, D). This selectivity may arise from the activation of the TLR4–MyD88 axis, but phagocytosis of tumor cells remained limited due to persistent CD47-SIRPα "do not eat me" signaling [9, 35].

IL-1β and iNOS are well-established biomarkers of macrophage activation [36,37,38]. To evaluate the immunomodulatory effects of the four WCP fractions, this study quantified the expression levels of IL-1β and iNOS ( a rate-limiting enzyme for NO synthesis encoded by the Nos2 gene), in RAW264.7 macrophages following treatment with WCP. As shown in Fig. 4C, all four WCPs significantly upregulated the expression of the Il1b and Nos2 genes in a dose-dependent manner (P < 0.05; Fig. 4E, 4G). This transcriptional activation correlated with increased nitric oxide (NO) and IL-1β (Fig. 4F, 4H). Collectively, these results demonstrate that all WCP fractions effectively promote macrophage activation.

Differential activation of MAPK/NF-κB signaling pathways

The phosphorylation of extracellular signal-regulated kinase (ERK), P38 mitogen-activated protein kinase (P38), and c-Jun N-terminal kinase (JNK) activates the MAPK cascade. Phosphorylation of the nuclear factor κB subunit p65 (p65) triggers its nuclear translocation, thereby initiating NF-κB signaling. The MAPK/NF-κB signaling pathway collectively drives the expression of pro-inflammatory genes in activated immune cells [8].

Although all four WCP fractions have the potential to enhance macrophage activation, the underlying mechanisms remain unclear. To address this issue, this study investigated alterations in the MAPK/NF-κB signaling pathway, which is a critical regulator of macrophage activation. As shown in Fig. 5A–D, all WCP fractions significantly increased the phosphorylation ratios of JNK (p-JNK/JNK) and ERK (p-ERK/ERK) in a dose-dependent manner (P < 0.05), indicating consistent activation of these MAPK subpathways. However, differential effects were noted for P38 and p65 (Fig. S3). Among them, WCP-40 and WCP-60 significantly increased P38 phosphorylation (P < 0.05, Fig. 5B). WCP-20, WCP-60, and WCP-80 significantly enhanced p65 phosphorylation (P < 0.05, Fig. 5E, F).

Fig. 5

WCP upregulates the expression of MAPK/NF-κB signaling proteins in RAW 264.7 cells . A–D MAPK pathway proteins. E, F NF-κB pathway proteins. Results were expressed as mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 compared to the NC group

To delineate the functional contributions of key signaling nodes, this study employed selective pharmacological inhibitors: PD98059 (ERK), SP600125 (JNK), SB203580 (P38), and BAY11-7082 (p65). Strikingly, WCP-20-induced macrophage activation, reflected by phagocytosis and Il1b and Nos2 expression, was suppressed by co-treatment with ERK, JNK, or NF-κB inhibitors (P < 0.05, Fig. 6A). This confirms that WCP-20 requires ERK, JNK, and p65 signaling to exert its immunostimulatory effects. . In parallel, WCP-40-driven activation was inhibited by suppressing ERK, JNK, and P38 (P < 0.05, Fig. 6B). WCP-60-mediated responses were significantly reduced following the inhibition of ERK, JNK, P38, or p65 (P < 0.05, Fig. 6C). WCP-80-dependent activity specifically relied on JNK and p65 signaling (P < 0.05, Fig. 6D). Collectively, all fractions activated MAPK/NF-κB signaling, though each of them displayed distinct mechanistic dependencies.

Fig. 6

WCP  activates RAW 264.7 cell via the MAPK/NF-κB signaling pathways. A WCP-20 activated the ERK, JNK, and p65 proteins. B WCP-40 activated the P38, ERK, and JNK proteins. C WCP-60 activated the P38, ERK, JNK, and p65 proteins. D WCP-80 activated both JNK and p65. Results were expressed as mean ± SD (n = 3). ^#P < 0.05, ^##P < 0.01, compared to the NC group. ^*P < 0.05, ^**P < 0.01, compared to the WCP group

Correlation analysis of physicochemical properties and immune activity

In this study, four WCP fractions were obtained through a graded ethanol precipitation process, and all enhanced macrophage activity with different strengths, suggesting potential structure-dependent effects. This study conducted a preliminary assessment of correlation, focusing on the relationships between structural features and  bioactivity. Cluster analysis revealed a closer physicochemical similarity between WCP-20 and WCP-40, as well as between WCP-60 and WCP-80 (Fig. 7A), indicating distinct structural groupings.

Fig. 7

Correlation analysis of physicochemical properties and immunomodulatory activity. A Structural cluster heatmap of WCPs; B Structural correlation coefficient matrix; C Bioactivity-correlated structural cluster heatmap; D Correlation network heatmap between monosaccharide composition and immunomodulatory activity; E Correlation network heatmap between secondary structure and immunomodulatory activity; F Correlation network heatmap between other physicochemical properties and immunomodulatory activity

In structural correlation analysis, MW positively correlated with Dh (Fig. 7B), indicating that higher-MW polysaccharides possess larger Dh in solution, which readily form colloidal solutions and demonstrate the Tyndall effect. Mannose content negatively correlated with glucose content, MW, and Dh, suggesting that mannose-rich WCPs adopt smaller conformations compared to glucose-rich WCPs. The contents of galactose, glucose, and mannose, especially galactose, were significantly correlated with specific optical rotation. This suggests that the monosaccharide composition has a strong influence on optical rotation. Furthermore, a strong correlation exists between MW and crystallinity. Together, these data reveal strong interconnections among the physicochemical properties of WCPs, which may underlie their biological functions.

As illustrated in Fig. 6, WCPs modulated immune responses via the MAPK/NF-κB pathway, which was further validated through the use of inhibitors. To elucidate the correlations underlying this bioactivity, this study integrated structural parameters with immunological readouts (Fig. 7C). Positive correlations were observed between glucuronic acid, mannose, galactose, and helix content, as well as between ERK, NO, p65, IL-1β, and JNK. By contrast, crystallinity, random coil content, glucose proportion, and MW correlated negatively with ERK, NO, p65, and IL-1β, but positively with phagocytosis and Nos2. Notably, significant correlations were observed between specific monosaccharides and proteins: glucose and galactose with JNK, mannose with Il1b, and glucuronic acid with ERK (P < 0.05, Fig. 7D). Regarding secondary structure, Nos2 exhibited significant positive correlations with helix, antiparallel, parallel, beta-turn, and random coil content (P < 0.05, Fig. 7E). Additionally, the Dh exhibited a positive correlation with ERK (P < 0.05, Fig. 7F). These results suggest that the bioactivity of WCP is strongly influenced by its physicochemical properties. However, the causal mechanisms behind these correlations need  to be explored with additional experimental validation .

Discussion

Summary of research results

Through stepwise ethanol precipitation, this study achieved the isolation of structurally differentiated WCP fractions, which were subsequently shown to modulate immune responses via disparate mechanisms. This study demonstrated that WCP fractions (WCP-20 to WCP-80) possessed MW-dependent physicochemical characteristics: higher-MW fractions (WCP-20/-40) were glucose-rich with triple-helical conformations and superior thermal stability, while lower-MW fractions (WCP-60/-80) displayed higher galactose and mannose content, branching density, and optical activity. Crucially, all WCPs activated macrophages through the MAPK/NF-κB pathway but engaged divergent signaling nodes, specifically involving ERK/JNK/p65 (WCP-20), P38/ERK/JNK (WCP-40), P38/ERK/JNK/p65 (WCP-60), and JNK/p65 (WCP-80), revealing structure-dependent immunomodulatory mechanisms. Correlation analyses further linked bioactivity to monosaccharide composition (e.g., glucose and galactose content with JNK activation) and secondary structures (e.g., helical motifs with Nos2 induction). These findings offer the first evidence that ethanol-fractionated WCPs precisely tune immune responses through MW-governed structural hierarchies. This work establishes a comprehensive framework for developing standardized C. sinensis-derived immunotherapeutics with tailored efficacy.

Analysis of physical and chemical properties

Previous MW analyses of Cordyceps polysaccharides conducted by our group revealed a broad MW distribution profile [7], consistent with other reports [39, 40]. Such structural complexity and wide MW range hinder the chromatographic isolation of homogeneous polysaccharides from C. sinensis [41]. Graded ethanol precipitation overcomes this limitation by modulating hydrogen bonding and van der Waals interactions in polysaccharide solutions, allowing for the rapid fractionation of structurally distinct polysaccharide subsets based on MW [12]. Accordingly, this study employed ethanol fractional precipitation (20%, 40%, 60%, and 80%) to isolate four polysaccharide fractions with differing MW: WCP-20 (5.4 × 10⁴ kDa), WCP-40 (3.6 × 10² kDa), WCP-60 (38 kDa), and WCP-80 (23 kDa). Comparative analysis revealed MW discrepancies between polysaccharides isolated at identical ethanol concentrations: WCP-80 (23 kDa) versus CSP1a (15.7 kDa) precipitated at 80% ethanol [28] and WCP-40 versus CCP (433.8 kDa) precipitated at 40% ethanol [42]. These discrepancies may stem from differences in commercial grades and geographical origins of C. sinensis specimens [43].

Monosaccharide composition analysis is crucial for elucidating the structure of polysaccharides. Earlier work indicated that WCP primarily consists of glucose, mannose, and galactose [44,45,46]. In this study, trace amounts of glucuronic acid were detected in all four WCP fractions. Interestingly, higher ethanol concentrations correlated with increased mannose and galactose content, while glucose levels declined substantially. These ethanol-mediated compositional trends aligned with prior chromatographic analyses [14]. Additionally, all WCP samples contained minor protein contamination but no nucleic acids. Although nucleic acids are known bioactive constituents of C. sinensis, it may be hydrolyzed into small molecules during high-temperature  extraction [47], and likely removed during dialysis. UV-Vis and FT-IR spectra confirmed the presence of trace proteins in all four WCP samples, despite the use of the Sevag method to eliminate free proteins from the polysaccharide solution. This outcome may be attributed to the intrinsic presence of glycoproteins in Cordyceps mycelia, these glycoproteins are known to possess antioxidant and immunomodulatory activities [48, 49]. Due to limited polysaccharide yield, column chromatography was not used for further purification. Future studies should test this hypothesis and further investigate the immunomodulatory potential of native glycoproteins in wild C. sinensis.

GPC analysis indicated that all four WCPs showed unimodal distributions, with their MW demonstrating a sharp decrease as the ethanol concentration increased, consistent with prior reports [15, 26, 50]. This result demonstrate that ethanol concentration directly modulates polysaccharide MW, enabling precise control for tailored applications. All polysaccharide solutions also exhibited Tyndall effects, confirming their colloidal nature. DLS analysis similarly revealed unimodal profiles in aqueous solution, with mean particle diameters positively correlating with MW.

The triple-helix conformation of polysaccharides exerts a critical influence in regulating immunomodulatory activity [33]. Existing evidence indicates that glucans with MW above 90 kDa readily form triple helices, while those below 50 kDa do not, highlighting MW dependency [33]. In the characterization of secondary structure by CD spectroscopy, higher-MW fractions (WCP-20/-40) predominantly exhibited antiparallel, parallel, beta-turn, and random coil signatures, while WCP-60 and WCP-80 displayed primarily helical motifs. In the Congo red assay assessing tertiary structure, WCP-20 and WCP-40 exhibited distinct triple-helix conformations. Conversely, WCP-60 and WCP-80 likely lack the triple-helix conformation and instead adopt  single- or double-helix structure. The iodine–potassium iodide (I₂–KI) test indicated that WCP-20 had fewer branched chains, while WCP-40, WCP-60, and WCP-80 exhibited a relatively higher branching density. Based on monosaccharide composition, WCP-20 is predominantly glucose, whereas WCP-40/-60/-80 are rich in mannose and galactose. Two previous studies isolated a glucan [51] and a galactomannan [41] from C. sinensis. The proposed structures suggest that the glucan is sparsely branched, while the galactomannan is highly branched, which is consistent with the branching patterns inferred from our monosaccharide data. Additionally, a low branching ratio is essential for triple-helix formation [52], supporting the Congo red assay results.

In the physicochemical characterization of solid-state materials, SEM imaging revealed that all four Cordyceps polysaccharides exhibited composite morphologies, including sheet-like, filamentous, and rod-like structures, consistent with observations reported in other studies [7, 41]. Notably, the proportion and surface area of sheet-like structures increased with MW, while filamentous features decreased, likely due to reduced branching density. Thermal stability was slightly higher in WCP-20/-40 than in WCP-60/-80, possibly because the latter contains abundant short chains and side branches that form looser packing, reducing thermal resistance [53]. The crystallinity of polysaccharides is primarily influenced by multiple factors, including molecular structure (linear/branched), hydrogen bond strength, and extraction/processing methods [34]. In this study, all four polysaccharides exhibited low crystallinity, which is consistent with other studies [34]. Intriguingly, the overall XRD diffraction peak intensity of Cordyceps polysaccharides increased as the MW decreased (Fig. 2I), indicating that their crystalline properties are regulated by factors such as MW.

Analysis of immune activity and signaling pathways

Previous studies from our research group demonstrated that WCP enhanced innate immunity by increasing the proportion of macrophages in the spleens of H22 hepatocellular carcinoma-bearing mice. In vitro experiment also demonstrated that WCP could enhance oxidized low-density lipoprotein-induced macrophage efferocytosis via PPAR-γ activation [54]. The TLR4/MAPK/NF-κB pathway regulates immune responses by coordinating the production of pro-inflammatory cytokines and the activation of immune cells, where MAPK cascades (ERK, JNK, and P38) synergize with NF-κB to amplify inflammatory signaling and shape innate and adaptive immunity [55]. Although prior studies isolated two polysaccharides from fermented C. sinensis and reported MAPK/NF-κB activation [56, 57], they neither used pathway inhibitors nor correlated physicochemical properties and bioactivity.

In this study, RT-qPCR, Western blot, and pathway inhibitors were adopted to delineate the role of MAPK/NF-κB signaling in WCP’ immunomodulation. Differential activation was observed: WCP-20 primarily phosphorylated ERK, JNK, and p65; WCP-40 enhanced P38, ERK, and JNK phosphorylation; WCP-60 augmented all MAPK components plus p65; WCP-80 specifically activated JNK and p65. Co-treatment with specific inhibitors (ERK, JNK, P38, and NF-κB) and polysaccharides in macrophages significantly reduced phagocytic activity and the expression of Nos2 and Il1b, confirming the essential role of MAPK/NF-κB signaling in WCP immunomodulation (Fig. 8).

Fig. 8

WCPs activate the MAPK/NF-κB signaling pathway, thereby inducing macrophage production of IL-1β, iNOS, and NO

Correlation analysis

Correlation analysis effectively examined relationships between physicochemical properties and bioactivities. Its application to polysaccharides aids the rational design of functional carbohydrate products. For instance, Zhang et al. obtained four Zizania latifolia polysaccharide fractions via ethanol-gradient precipitation and used Pearson correlation to show that negative charge density, MW, arabinose and galactose content underlie antioxidant, moisturizing, and whitening activities [58]. Cs-HK1 is a fungus isolated from C. sinensis; Huang et al. [59] applied gradient ethanol precipitation to Cs-HK1 fermentation broth and observed that high-MW polysaccharide-rich fractions precipitated first, followed by lower-MW protein-enriched fractions. The protein-rich fractions exhibited the most pronounced antioxidant activity. In addition, Wang et al.[44] found that total phenolic content paralleled the ferric reducing ability exhibited by Cordyceps polysaccharides. Critically, early investigations failed to establish correlative relationships of these phenomena [44, 59]. This study identifies significant correlations between polysaccharide properties and immunostimulatory effects: glucose and galactose levels strongly correlate with JNK phosphorylation; glucuronic acid content and Dh associate closely with ERK activation; and Nos2 expression correlates with secondary structure. Future studies should clarify the mechanisms behind these correlations.

Comparative analysis of wild and fermented Cordyceps polysaccharides in the literature

A recent review notes that seven fungal species have been isolated from C. sinensis for polysaccharide extraction and identification [18]. Although these fermented mycelia are intended to mimic natural C. sinensis, substantial differences in growth conditions, nutrient sources, and life cycles result in structural and functional disparities in their polysaccharides. Previous research using saccharide mapping revealed that both natural and cultured Cordyceps polysaccharides contain 1,4-α/β-D-glucosidic and 1,4-α-D-galactosidic linkages. However, cultured strains may exhibit structural divergence due to strain or process differences [60, 61]. Wang et al.[44] demonstrated that polysaccharides from C. sinensis possess higher MW, simpler glucose-dominant compositions, and relatively uniform → 4)-Glcp-(1 → backbones, while cultured mycelia polysaccharides exhibit lower MW, more diverse monosaccharide profiles, and markedly heterogeneous glycosidic linkages.

Wild and fermented Cordyceps polysaccharides share similarities and differences in various bioactivities. For example, in vitro antioxidant assays show comparable DPPH and hydroxyl radical scavenging capacity between sources [44]. In addition, Yi et al.[62] revealed that natural C. sinensis (CS) polysaccharides contain higher proportions of immunomodulatory 1,4-Glcp glycosidic linkages and a dominant MW range, whereas polysaccharides extracted from fermented mycelia (CSF) display broader Mw distributions, higher glucose content, and variable 1,4-Glcp levels. Bioassays demonstrated CS polysaccharides strongly enhance macrophage phagocytosis, NO/TNF-α production, and IL-6 secretion at low doses, while only certain CSF batches (e.g., CSF4) mimic this activity [62]. Correlation analyses further linked glucose content, MW, and glycosidic bond types to immunomodulatory effects [62], consistent with aspects of this study. Distinctively, by using inhibitors, this work directly implicated the MAPK/NF-κB pathway in WCP immunostimulation and correlated WCP physicochemical properties with immune signaling protein expression.

Significance of the study

In this study, natural C. sinensis was collected from farmers in its genuine producing region. Using a non-hazardous graded ethanol precipitation technique, this study isolated four WCP with distinct structural features. Through chemical and biological approaches, this study elucidated the molecular mechanism by which WCP activates macrophages to exert immunostimulatory effects, thereby clarifying the structural basis of polysaccharides as the bioactive components responsible for C. sinensis' immunomodulatory properties. These findings provide a scientific rationale for the clinical application of C. sinensis in treating immune-related disorders in TCM. Furthermore, by linking physicochemical properties to immune activity, this study may enhance the medicinal value of wild-harvested C. sinensis, supporting sustainable economic development in Tibetan communities.

Research prospect

Cordyceps polysaccharides show promise for its potential immunomodulatory, anti-inflammatory, and anticancer effects. Although preliminary structural characterization was conducted, a complete elucidation  of chemical structures—including saccharide residue composition and glycosidic linkage patterns—requires advanced techniques, such as nuclear magnetic resonance  spectroscopy and methylation analysisto achieve comprehensive structural resolution Moreover, the current findings derived from in vitro cellular models require validation through in vivo animal studies to assess their immunomodulatory efficacy and therapeutic potential. Given the complex absorption kinetics of these polymeric carbohydrates, a pressing research gap exists regarding their pharmacokinetic behavior. Fluorescent tracer methodologies could reveal whether macrophage-mediated endocytosis improves systemic absorption and immunoregulation. This effort will advance ethnopharmacological understanding and promote innovative biopharmaceutical development.

Conclusion

This study demonstrated that Cordyceps polysaccharides (WCP-20, WCP-40, WCP-60, WCP-80) exhibited distinct MW-dependent physicochemical properties and immunomodulatory activities. Specifically, WCP precipitated with lower ethanol concentrations exhibited higher MW and the highest glucose content, while those obtained at higher ethanol concentrations showed smaller MW and greater galactose and mannose proportions. Cluster analysis revealed a greater similarity in physicochemical characteristics between WCP-20 and WCP-40, as well as between WCP-60 and WCP-80. Correlation analysis revealed intrinsic interrelationships among the physicochemical properties of WCP. In activity assessment assays, all four WCPs stimulated the production of IL-1β, iNOS, and NO in macrophages by activating the MAPK/NF-κB signaling pathway. Pathway inhibitor assays revealed polysaccharide fraction-specific modulation of MAPK/NF-κB pathway protein, indicating structure-dependent pathway activation. The correlation analysis also confirmed strong links between immunostimulatory effects and physicochemical properties, particularly monosaccharide composition and secondary structure. Together, these insights connect polysaccharide properties to immune function, offering a roadmap for developing functional products.