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Article

Purification, Structural Characterization, and Immunomodulatory Activity of Polysaccharides from Cinnamomum cassia

by
Jinya Dong
1,†,
Peng Zhang
2,†,
Subramanian Palanisamy
2,3,
Huajie Yin
1,
Qiyuan Zhang
1,
Chongye Fang
1,
SangGuan You
2,3,* and
Yunfei Ge
1,2,*
1
College of Food Science and Technology, Yunnan Agricultural University, Kunming 650201, China
2
Department of Marine Food Science and Technology, Gangneung-Wonju National University, Gangneung 25457, Republic of Korea
3
East Coast Research Institute of Life Science, Gangneung-Wonju National University, Gangneung 25457, Republic of Korea
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Foods 2026, 15(5), 927; https://doi.org/10.3390/foods15050927
Submission received: 31 January 2026 / Revised: 25 February 2026 / Accepted: 1 March 2026 / Published: 6 March 2026
(This article belongs to the Special Issue The Role of Functional Foods, Nutraceuticals, and Food Supplements in Health Promotion and Disease Prevention)
Browse Figures
Versions Notes

Abstract

In this manuscript, we report the successful purification of two polysaccharide fractions (F1 and F2) from Cinnamomum cassia (C. cassia). Their chemical composition analysis revealed carbohydrates (54.8–61.1%), sulfates (8.1–9.5%), proteins (4.8–8.0%), and uronic acids (3.7–3.9%), with molecular weights ranging from 46.1 to 2919.1 kDa. Methylation analysis indicated that the highly active F2 fraction possesses a main chain of (1 → 4)-linked glucose, with minor side chains of (1 → 3)- and (1 → 5)-linked arabinose or (1 → 6)-linked glucose, and terminal glucose/arabinose residues. In vitro experiments demonstrated that F2 significantly enhanced nitric oxide and cytokine (TNF-α, IL-1β, IL-6, IL-10) production in RAW264.7 macrophages through activation of NF-κB and MAPK signaling pathways, exhibiting stronger immunomodulatory activity than F1. These results provide evidence that C. cassia polysaccharides, particularly F2, possess promising potential as natural immunostimulants for functional food or therapeutic applications.

1. Introduction

Cinnamomum cassia (C. cassia), a perennial tree that is part of the Lauraceae family, stands out as a notable tropical species appreciated for its various uses [1]. Its raw materials, primarily derived from bark and leaves, are extensively utilized as a flavoring additive in food seasonings and play a significant role in traditional medicine [2,3]. Furthermore, its compounds are leveraged in industrial sectors for composite materials, and its essential oil extracts have shown promising effects on poultry immunity and microbiology [4]. The synergistic combination of C. cassia essential oil with chitosan nanofibers has also yielded high-performance active packaging films, demonstrating altered mechanical properties and water vapor permeability [5]. C. cassia exhibits a variety of pharmacological properties, which encompass antitumor, anti-inflammatory, analgesic, antiobesity, antibacterial, antiviral, cardiovascular protective, cytoprotective, neuroprotective, immunomodulatory, and effects that inhibit tyrosinase activity [2].
Recently, plant polysaccharides have garnered significant research interest as immune adjuvants and functional food ingredients [6]. This attention stems from their desirable characteristics, including low toxicity, high safety, and multi-target action as natural immunomodulators. Polysaccharides’ intricate immunomodulatory activity depends heavily on their diverse structural characteristics, which encompass factors such as monosaccharide types, glycosidic bonds, molecular size, branching degree, and spatial conformation [7]. Numerous studies have elucidated the diverse immunomodulatory mechanisms of plant polysaccharides. For instance, Panax notoginseng polysaccharides stimulate immunity by enhancing spleen lymphocyte proliferation, nitric oxide (NO) and cytokine (tumor necrosis factor-α, interleukin-2, interleukin-10, and interferon-γ) production, and increasing phagocytosis in peritoneal macrophages and Toll-like receptor 2 (TLR2) expression [8]. Additionally, Ganoderma leucocontextum polysaccharide GLP-3, rich in glucose, modulates immune responses by activating mitogen-activated protein kinase (MAPK), phosphatidylinositol-3-kinase (PI3K)/Akt, and nuclear factor-κB (NF-κB) [9]. Immunomodulatory effects have also been demonstrated for polysaccharides derived from Rehmannia glutinosa [10], Cistanche deserticola [11], and Millettia Speciosa [12]. Considering these multifaceted benefits, a comprehensive study of polysaccharide structure and biological function within functional foods is essential.
C. cassia is rich in various active components, including essential oils, phenolic compounds, flavonoids, lignans, diterpenoids, coumarins, and polysaccharides [13,14]. Driven by a burgeoning interest in natural macromolecules, C. cassia polysaccharides have increasingly captivated researchers. Previous studies have successfully extracted C. cassia polysaccharides through methods like water extraction–alcohol precipitation and microwave-assisted extraction, demonstrating their in vitro antioxidant and hypoglycemic activities [15,16]. As well as polysaccharides, other constituents of C. cassia essential oil have been shown to have antibacterial, antioxidant, and immunomodulatory properties [17], while its polyphenols have been reported to improve macrophage activity [2]. However, comprehensive and systematic characterization of C. cassia polysaccharide structural features (e.g., monosaccharide composition, glycosidic bond types, and molecular weight distribution) and their underlying immunomodulatory mechanisms remains limited. In this study, we employed current systematic polysaccharide research methods to extract, separate, purify, and structurally characterize polysaccharides from C. cassia. Concurrently, we performed in vitro assays to evaluate their antioxidant and immunomodulatory activities, providing a solid foundation for developing C. cassia polysaccharides into valuable foods and pharmaceuticals in the future.

2. Materials and Methods

2.1. Materials and Reagents

C. cassia powder was purchased from Hagimex JSC (Hanoi, Vietnam). it was derived from the dried stem bark and ground to a fine powder. Cell culture media (RPMI-1640 and α-MEM) were procured from Lonza (Walkersville, MD, USA). Lipopolysaccharide (LPS), along with Griess reagent and standards of monosaccharides such as L-rhamnose, L-arabinose, D-mannose, D-glucose, and D-galactose, were obtained from Sigma-Aldrich (St. Louis, MO, USA). The remaining chemical reagents utilized in this research were of analytical quality.

2.2. Extraction and Purification of C. cassia Polysaccharides

Pre-treatment of the fine powder of C. cassia (70 g) involved soaking it overnight in 500 mL of 80% ethanol for 12 h at 25 ± 1 °C with gentle stirring at ambient temperature to remove lipids, pigments, and other low-molecular-weight constituents. After washing the powder with diethyl ether, the ethanol–insoluble residue was air-dried at 25 ± 1 °C in a fume hood. The decolorized residue was dispersed in 400 mL of distilled water and extracted at 65 °C for 2 h with continuous stirring to maximize polysaccharide solubility and yield. The extract was centrifuged at 10 °C (5000× g) for 10 min to minimize thermal degradation and preserve biological activity. The resulting supernatant was concentrated under reduced pressure, and cold 96% ethanol was added until the final ethanol concentration reached 70% (v/v) to precipitate crude polysaccharides. The precipitate was collected by centrifugation, sequentially washed with ethanol and acetone, and dried at ambient temperature.
Crude polysaccharides were separated employing a DEAE Sepharose Fast Flow column (GE Healthcare BioScience AB, Uppsala, Sweden) at a flow rate of 1.5 mL/min and under room temperature conditions. A solution was prepared by dissolving 250 mg of crude polysaccharides in 10 mL of distilled water, which was then stirred for 15 min at a temperature of 65 °C. After loading the filtered sample solution onto a column with distilled water, a linear gradient of NaCl (0.5–2 mol/L) was applied. Using the phenol-sulfuric acid method [18], fractions containing carbohydrates were identified. Obtain two polysaccharide fractions, F1 and F2, the identified fractions were pooled, dialyzed, and freeze-dried.

2.3. Structural Analysis of C. cassia Polysaccharides

2.3.1. Chemical Composition and Monosaccharide Composition Analysis

Neutral sugar levels in the isolated polysaccharide preparations were measured using the phenol–sulfuric acid assay, and absorbance was measured at 490 nm using D-glucose as the calibration standard [19]. Protein concentration was determined using the Lowry assay, involving alkaline copper treatment followed by reaction with Folin–Ciocalteu reagent, with bovine serum albumin (BSA) used as the reference standard and absorbance measured at 750 nm [20]. Sulfate content was quantified using the Dodgson and Price potassium sulfate method, based on the turbidimetric measurement of barium sulfate formation after reaction with barium chloride–gelatin reagent [21]. Uronic acids were analyzed by the sulfonamide/m-hydroxydiphenyl assay, and absorbance was recorded at 525 nm using uronic acid as the standard [22].
Polysaccharide samples (2 mg) were precisely weighed, and 4 M trifluoroacetic acid (TFA) was used for hydrolysis in sealed glass tubes at 100 °C for 6 h. Under a nitrogen stream, TFA was completely evaporated after hydrolysis. The resulting monosaccharides were reduced with 10 mg sodium borodeuteride (NaBD4) at room temperature and subsequently acetylated to form alditol acetates. Gas chromatography–mass spectrometry (GC–MS; Agilent 6890N/5973 MSD, Santa Clara, CA, USA) was utilized to analyze the derivatives, employing an HP-5MS capillary column with dimensions of 30 m × 0.25 mm × 0.25 μm. Helium was used as the carrier gas at a constant flow rate of 1.2 mL/min. Samples were injected in split mode with a split ratio of 10:1. The oven temperature was programmed from 160 °C to 280 °C at a rate of 5 °C/min, with a final hold at 280 °C for 10 min.

2.3.2. Molecular Weight Analysis

Lyophilized polysaccharides were dissolved in distilled water to 2 mg/mL and briefly microwaved at 75 °C for 30 s to facilitate dissolution [23]. Approximately 20 μL of each solution was injected into a high-performance size-exclusion chromatography setup. Separation was carried out on a TSK G5000PW column (7.5 × 600 mm; Toso Biosep, Montgomeryville, PA, USA) using 0.15 mol/L NaNO3 containing 0.02% NaN3 as the mobile phase at a flow rate of 0.4 mL/min, coupled with a multi-angle laser light scattering detector (HELEOS, Wyatt Technology Corp., Santa Barbara, CA, USA) and an index detector (Waters 2414), forming an HPSEC–MALS–RI configuration. Weight-average molecular mass (Mw) and radius of gyration (Rg) were determined using ASTRA software version 5.3 (Wyatt Technology Corp.).

2.3.3. Glycosidic Bond Analysis

Glycosidic linkages were elucidated through methylation analysis, a method derived from the protocol of Ciucanu and Kerek [24]. A sample of polysaccharides was suspended in dimethyl sulfoxide, and 20 mg of NaOH and 0.3 mL of CH3I were added under a nitrogen atmosphere. Methylation was performed by maintaining the reaction at room temperature for 45 min. Following methylation, the derivatives were hydrolyzed in 4 M trifluoroacetic acid at 100 °C for 6 h, reduced in distilled water with sodium NaBD4, and finally acetylated with acetic anhydride at 100 °C. Finally, partially methylated alditol acetates (PMAAs) were analyzed by GC–MS with helium as the carrier gas maintained at 1.2 mL/min. Absolute sugar configurations were determined by GLC following derivatization to acetylated (R)-2-methylheptyl glycosides [25].

2.4. Immunomodulatory Activity of C. cassia Polysaccharides

2.4.1. Cell Proliferation and NO Release Assays

The immunomodulatory effects of polysaccharides were evaluated in RAW 264.7 macrophages (ATCC, Rockville, MD, USA). Cells were cultured in RPMI-1640 media enriched with penicillin (100 U/mL), streptomycin (100 μg/mL), and 10% fetal bovine serum at 37 °C in a 5% CO2 atmosphere. Cells were seeded at a density of 1 × 106 cells/well (100 μL per well) and cultured for 24 h. After which, polysaccharide samples were added at varying concentrations (ranging from 25 to 100 μg/mL) for an additional 24 h. Cell proliferation was determined by adding 20 μL of WST-1 reagent to each well (DoGenBio Co., Ltd., Seoul, Republic of Korea). Absorbance was measured at 450 nm with a microplate spectrophotometer. After polysaccharide treatment, cell supernatants were collected in separate 96-well plates for nitric oxide (NO) production assays. Griess reagent was introduced into the wells, and the absorbance was recorded at a wavelength of 540 nm [26].

2.4.2. Cytokine Gene Expression Analysis

RAW 264.7 cells at a concentration of 1 × 106 cells/mL (100 μL per well) were subjected to incubation with 50 µg/mL of either polysaccharide or liposomes for a duration of 18 h. Total RNA was obtained from the cells that were treated with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) in accordance with the supplier’s guidelines. The purified RNA was then reverse-transcribed to cDNA employing oligo(dT)20 primers and Superscript III reverse transcriptase, following the recommended protocol. RT-qPCR assays were carried out on a real-time thermocycler using the FastStart DNA Master TB Green II kit (Takara Bio, Shiga, Japan) together with primer pairs targeting cytokine genes (Table S1). Relative gene expression was determined through the 2−ΔΔCt method, utilizing β-actin as the reference internal gene.

2.4.3. NF-κB and MAPKs Signaling Pathway Analysis

RAW264.7 cells were exposed to C. cassia polysaccharide (50 µg/mL) at 37 °C for 18 h. After treatment, cells were harvested and subsequently lysed in ice-cold RIPA buffer, which contained 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate (SDS), along with a mixture of protease and phosphatase inhibitors (Abcam, Cambridge, UK). Protein levels were measured with the Pierce BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA). Cell lysates (30 μg for each lane) were subjected to electrophoresis on a 10% SDS-polyacrylamide gel (SDS-PAGE) and then transferred onto polyvinylidene difluoride (PVDF) membranes. Membranes were incubated for 2 h at room temperature in Tris-buffered saline containing 5% nonfat milk. Following overnight exposure to primary antibodies recognizing phospho-NF-κB p65, phospho-JNK, phospho-ERK, and phospho-p38 (Abcam, Cambridge, UK), membranes were treated with secondary antibodies labeled with HRP for 1 h at 4 °C. Target proteins were visualized by ECL in accordance with the supplier’s guidelines. Visualization of protein bands was performed with a Bio-Rad Image Analysis System (Bio-Rad Laboratories, Hurley, CA, USA), and protein expression was conducted using Quantity One software (version 4.6, Bio-Rad, USA).

2.4.4. Flow Cytometry Analysis

RAW 264.7 cells were seeded at 1 × 106 cells/well (2 mL per well) and treated with 50 μg/mL of C. cassia polysaccharide for 24 h at 37 °C in 5% CO2. After treatment, cells were harvested and prepared for flow cytometry to assess changes in cell surface marker expression. Incubation at 4 °C in the dark with fluorochrome-conjugated antibodies: anti-CD40-APC (Clone 1C10) and anti-CD11b-PE (Clone M1/70) for 30 min was then used. Flow cytometry was conducted on a CytoFLEX flow cytometer, and the resulting data were analyzed using manufacturer-provided software (Beckman Coulter Limited, High Wycombe, UK), which was supplied.

2.5. Statistical Analysis

All experiments were independently repeated three times (n = 3), and data are expressed as mean ± standard deviation (SD). Statistical analyses were performed using SPSS (version 16.0; SPSS Inc., Chicago, IL, USA). The differences among the groups were evaluated using a one-way analysis of variance (ANOVA), followed by post hoc comparisons that utilized Duncan’s multiple range test. A statistical significance level of p < 0.05 was established.

3. Results and Discussion

3.1. Yield and Chemical Composition of Polysaccharides Extracted from C. cassia

The crude polysaccharide from C. cassia was isolated using a hot water extraction method followed by ethanol precipitation. The yield and chemical composition of these polysaccharides are presented in Table 1. In this study, the crude polysaccharide from C. cassia was obtained at a yield of 42.4%. Its chemical composition (%, w/w) primarily consisted of carbohydrates (49.3 ± 3.8), along with protein (10.3 ± 0.5), sulfate (11.3 ± 1.8), and uronic acid (5.6 ± 0.3). Subsequently, the crude polysaccharide was further fractionated using DEAE-Sepharose fast-flow column chromatography. As depicted in Figure 1, two distinct fractions, designated F1 and F2, were observed with yields of 24.4% and 15.7%. F1 contained carbohydrates (54.8 ± 1.4), protein (4.8 ± 0.2), sulfate (8.1 ± 1.6), and uronic acid (3.9 ± 0.1), while F2 comprised carbohydrates (61.1 ± 6.0), protein (8.0 ± 0.1), sulfate (9.5 ± 1.1), and uronic acid (3.7 ± 0.7). The overall polysaccharide yield obtained by the hot water extraction method in this study (42.4%) was superior to that reported for polysaccharides isolated from the same species (13.48% ± 0.38%) using a microwave-assisted extraction method [16]. These differences in yield are likely attributable to differences in harvest sites, plant growth environments, and the specific extraction methodologies employed [27]. Higher purity polysaccharide preparations generally exhibit more defined bioactivity profiles, as residual proteins, pigments, or small molecules may cause non-specific immune stimulation or interference [28,29].

3.2. Monosaccharide Composition Analysis

GC-MS was used to determine the monosaccharide profiles of crude polysaccharides, F1, and F2 (Figure 2 and Table 2). The crude polysaccharide was primarily composed of glucose (94.7%), accompanied by arabinose (2.2%) and galactose (1.2%), with minor amounts of xylose (0.8%) and rhamnose (0.4%). Fraction F1 exhibited an even higher proportion of glucose (98.8%), along with trace amounts of arabinose (0.8%), xylose (0.3%), and rhamnose (0.1%). Notably, galactose was not detected in F1. In contrast, F2 primarily consisted of glucose (91.7%), followed by arabinose (3.5%) and galactose (2.8%), with trace amounts of xylose (1.0%) and rhamnose (0.9%). These findings align with previous reports, where glucose was identified as the major monosaccharide in polysaccharides derived from C. cassia [30]. Polysaccharides rich in glucose, especially with uniform backbone structures, can facilitate recognition by carbohydrate-binding receptors on immune cells. The presence of minor sugars such as arabinose and galactose can introduce branching and structural heterogeneity, potentially affecting receptor specificity and immune modulation [31]. In our data, the higher arabinose and galactose content in F2, compared with F1.

3.3. Molecular Weight Distribution of Polysaccharide Fractions

The molecular weight and distribution of polysaccharide samples were analyzed using HPSEC-MALLS-RI. As shown in the refractive index chromatograms and supported by the monosaccharide composition analysis (Figure 3 and Table 3), the crude polysaccharide and its fractions F1 and F2 exhibited multiple peaks. Specifically, the crude polysaccharide displayed three distinct peaks (Figure 3A), corresponding to weight-average Mw of 4321.3 kDa (Peak I), 214.1 kDa (Peak II), and 154.1 kDa (Peak III). In contrast, fractions F1 and F2 each exhibited two peaks in their respective RI chromatograms. For the F1 polysaccharide (Figure 3B), Mw values were determined to be 2919.1 kDa (Peak I) and 72.4 kDa (Peak II). Similarly, the F2 polysaccharide showed Mw values of 69.0 kDa (Peak I) and 46.1 kDa (Peak II) (Figure 3C). The Rg was determined as the root-mean-square distance of polymer segments from the molecular center of mass. In the crude polysaccharide, peaks I, II, and III exhibited Rg values of 60.2, 71.7, and 74.7 nm, respectively. Fraction F1 showed Rg values of 59.8 nm for peak I and 70.4 nm for peak II, whereas fraction F2 yielded 71.2 nm and 70.9 nm for peaks I and II, respectively. Furthermore, calculation of the SVg (scaling exponent relating Rg to Mw) values for the polysaccharide samples revealed that the crude polysaccharide exhibited the most compact molecular conformation, with an SVg value of 4.3 cm3/g. Conversely, F1 (SVg = 12.6 cm3/g) and F2 (SVg = 19.5 cm3/g) demonstrated looser structures and more extended molecular conformations. According to this study, the molecular weight of polysaccharides isolated from C. cassia was higher. Research indicates that larger molecular weights and more compact structures of polysaccharides are often associated with their solubility, viscosity, and ability to interact with targets [32]. However, these characteristics may also diminish the polysaccharides’ biological activity, thereby limiting their applications in immunomodulation, antioxidant effects, and anti-inflammatory therapies [33]. Conversely, polysaccharides with lower molecular weights often exhibit weaker intramolecular hydrogen bonding interactions, resulting in more free amino and hydroxyl groups. This configuration may facilitate swelling and enhance interaction with environmental molecules [34].

3.4. Effects of C. cassia Polysaccharides on Macrophage Activation

3.4.1. Cell Proliferation and Nitric Oxide Production

The innate immune system relies on macrophages, known for their responses to pathogens, viruses, bacteria, toxins, and other harmful stimuli [30]. LPS induced RAW264.7 macrophages are a widely utilized in vitro model to assess the immunomodulatory activity of natural polysaccharides [35]. The immunostimulatory activity of crude polysaccharides from C. cassia were assessed in RAW264.7 macrophages at concentrations of 25–100 μg/mL. Results demonstrated that crude extracts, F1, and F2 within this concentration range did not affect RAW264.7 cell viability and exhibited no cytotoxic effects on macrophages (Figure 4A).
Polysaccharides stimulate macrophages to release inflammatory mediators, including NO, ROS, TNF-α, and interleukin-1β (IL-1β) [36]. NO, a crucial signaling molecule involved in diverse pathophysiological functions, is rapidly released by activated macrophages to combat invading pathogens, fungi, or tumor cells [37]. Hence, measurement of nitric oxide is a pivotal metric for gauging the immunomodulatory effects of polysaccharide-derived compounds [38]. C. cassia polysaccharides were used to treat RAW 264.7 cells at varying concentrations to assess the production of NO. Samples elicited a concentration-dependent enhancement of NO production (Figure 4B). The amount of nitric oxide produced by cells treated with F2 polysaccharide (100 μg/mL) was higher than that produced by crude polysaccharide and F1 polysaccharide, but lower than that produced by cells treated with LPS (2 μg/mL). This observation aligns with prior reports on plant-derived polysaccharides, including those from Helicteres angustifolia L., where elevated macrophage NO production serves as a marker of immunomodulatory activity [30]. This suggests that F2 moderately stimulates NO secretion in RAW 264.7 macrophages, indicating its involvement in cellular immunomodulation.

3.4.2. Cytokine Production and Signaling Pathway Activation

The iNOS is the principal enzyme responsible for NO synthesis [39]. Its activation is tightly associated with NF-κB signaling and the regulation of cytokines, including the pro-inflammatory mediators TNF-α, IL-1β, and IL-6, as well as the anti-inflammatory interleukin-10 (IL-10) [39,40]. As illustrated in Figure 5A, iNOS mRNA expression levels in F2-treated RAW 264.7 cells were significantly elevated (p < 0.05) compared to the control. In contrast, crude polysaccharide and F1-treated cell groups exhibited relatively lower iNOS expressions. The results align with the NO production outcomes that were observed. Prior studies indicate that increased NO production in RAW 264.7 cells correlates with elevated iNOS expression [41].
Upon activation, macrophages secrete cytokines—low-molecular-weight proteins that orchestrate and modulate immune responses [42]. TNF-α involved in activating and promoting the growth of immune cells, thereby affecting the functions of T cells and B cells [43]. IL-1β and IL-6 are crucial for acute-phase immune responses and protein synthesis [44], while IL-10 primarily functions by suppressing inflammatory responses and maintaining immune tolerance [45]. In contrast to crude polysaccharides and F1 (Figure 5B–E), treatment with F2 polysaccharide significantly improved the expression of IL-1β, IL-6, IL-10, and TNF-α. (p < 0.05). Previous studies indicate that natural polysaccharides derived from Portulaca oleracea L. [46], Angelica gigas [47], and chia seed [35] also exert immunomodulatory effects by increasing NO production and cytokine secretion such as TNF-α and IL-6. This indicates that F2 moderately stimulates RAW 264.7 macrophages to release NO and proinflammatory factors, suggesting its potential immunomodulatory activity.
Research indicates that the MAPK and NF-κB pathways interact to finely tune macrophage function, playing a pivotal role in the immunomodulatory effects of various plant polysaccharides [48]. NF-κB, a central transcription factor in immune signaling pathways, requires the nuclear translocation of its p65 subunit for activation [49]. Within the MAPK family, ERK, JNK, and p38 are three major subfamilies that govern vital cellular processes including signaling, differentiation, inflammatory responses, immune responses, and apoptosis [50]. Specifically, ERK primarily controls cell proliferation, differentiation, and cell cycle regulation; JNK is central to cellular stress responses and inflammation; and p38 plays a key role in stress, inflammation, and apoptosis [51]. This study investigated whether NF-κB and MAPK signaling pathways participate in F2-induced macrophage activation by measuring the effects of F2 on the expression levels of phosphorylated p38, p65, JNK, and ERK proteins. It was found that F2 polysaccharides enhanced the phosphorylation levels of p38, JNK, ERK, and p65 in RAW 264.7 cells (Figure 5F and Figure S1). These findings suggest that F2 stimulates RAW 264.7 cells through the NF-κB and MAPK pathways.

3.5. The CD40 and CD11b Expression

In this study, the surface expression of CD40 and CD11b on RAW 264.7 cells were analyzed by flow cytometry following 24 h treatment with crude extract, F1, or F2 (50 µg/mL). CD40, a co-stimulatory molecule belonging to the TNF receptor superfamily, is primarily involved in macrophage activation through binding to its ligand CD40L on activated T cells [52]. CD11b plays a critical role in the adhesion, migration, phagocytosis, and regulation of inflammatory responses by macrophages, neutrophils, and NK cells [53]. Treatment with crude extract resulted in CD40 and CD11b expression levels of 35.73% and 22.85%. The F1 group showed slightly higher expression levels of CD40 (40.79%) and CD11b (24.78%). Notably, F2 induced a significantly greater increase in CD40 and CD11b expression compared with crude extract and F1 (p < 0.05), reaching 56.69% and 29.22% (Figure 6). Previous studies have demonstrated that the expression of CD40 and CD11b is markedly upregulated when macrophages are activated by microfibrils [54,55]. Engagement of CD40 drives B-cell activation and proliferation through NF-κB and MAPK signaling, thereby increasing the production of cytokines such as TNF-α, IL-6, and IL-10 [52,56]. CD11b indirectly modulates NF-κB activity and inflammatory responses by regulating cell adhesion and migration, as well as influencing IL-10 production [53]. Our data indicate that F2 stimulates RAW 264.7 macrophages via NF-κB and MAPK pathway activation. These pathways are activated under inflammatory conditions, where they synergistically increase the production of iNOS, NO, and multiple pro-inflammatory cytokines [57]. Consistent with this mechanism, F2 treatment significantly increased the levels of NO, iNOS, TNF-α, IL-1β, IL-6, and IL-10. Collectively, the findings indicate that F2 upregulates CD40 and CD11b on RAW 264.7 macrophages and promotes inflammatory mediator production by engaging NF-κB and MAPK pathways.

3.6. Glycosidic Linkages Analysis of F2 Fraction

Among the biochemical methods involved in identifying polysaccharide linkages and structural characteristics, methylation analysis is one of the crucial methods for determining complex carbohydrates’ linkage structures [58]. A methylation analysis of the F2 polysaccharide, which exhibits potent immunostimulatory activity, methylation analysis was performed. Eight derivatives were identified from the F2 polysaccharide (Table 4). The predominant derivative detected was 1,4,5-tri-O-acetyl-2,3,6-tri-O-methyl-glucose (92.3%), indicating that (1 → 4)-linked glucose residues constitute the major structural component. Other identified glucose linkages included terminal non-reducing glucosyl units (Glu → 1, 4.1%), (1 → 3,4)-linked glucose residues (1 → 3,4 Glu, 0.4%) indicating branching points, and minor proportions of (1 → 6)-linked glucose residues (1 → 6 Glu, 0.1%). Arabinose derivatives accounted for a smaller proportion, with the presence of terminal non-reducing arabinosyl units (Ara → 1, 1.2%), (1 → 5)-linked arabinose (1 → 5 Ara, 0.9%), and (1 → 3)-linked arabinose (1 → 3 Ara, 0.5%). Additionally, a derivative corresponding to (1 → 2,4)-linked glucitol (1 → 2,4 Glc, 0.5%) was detected. These findings indicate that F2 polysaccharide is primarily composed of a main chain of 1 → 4-linked glucose units, with branching occurs through (1 → 3,4) and (1 → 6) linkages. Arabinose also contributes to the structure, predominantly with (1 → 5) and (1 → 3) linkages, and terminal units of both glucose and arabinose are present. Glycosidic linkage patterns influence not only chain conformation but also degradation rates and receptor recognition. The predominance of β-(1 → 4) linkages in F2 may confer structural stability, while branching via (1 → 3,4) and (1 → 6) linkages could influence solubility and interaction with multiple receptor types. The minor arabinose linkages, such as (1 → 5) and (1 → 3), may also play supporting roles in modulating immune signaling.

4. Conclusions

In this study, polysaccharides isolated from C. cassia were extracted, purified, structurally characterized, and their immunomodulatory effects were investigated. There were two active fractions, F1 and F2, with different molecular weights and monosaccharide compositions. Analysis of glycosidic residues indicated that F2, the more active fraction, is dominated by glucose linked through (1 → 4) glycosidic linkages, with smaller amounts of arabinose and glucitol. Arabinose residues were connected through (1 → 5) and (1 → 3) linkages, while glucitol was linked via (1 → 2,4) bonds; the terminal residues were glucose and arabinose. In vitro assays demonstrated that F2 exhibited markedly stronger immunomodulatory activity than F1, significantly enhancing nitric oxide and cytokine production in RAW 264.7 cells through activation of the NF-κB and MAPK signaling pathways. Overall, these results provide valuable evidence supporting the potential of C. cassia polysaccharides, particularly the F2 fraction, as promising natural immunostimulants for functional food or therapeutic applications.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/foods15050927/s1, Table S1. Primers sequences used for real-time PCR. Figure S1. Phosphorylation levels of key proteins p65, JNK, ERK, and p38.

Author Contributions

J.D.: Writing—review & editing, Data curation, Conceptualization. P.Z.: Writing—original draft, Data curation, Conceptualization. S.P.: Writing—review & editing, Investigation. H.Y.: Investigation. Q.Z.: Investigation. C.F.: Formal analysis. S.Y.: Funding acquisition, Project administration, Resources. Y.G.: Writing—review & editing, Funding acquisition, Resources. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Foreign Talent Introduction Special Project, project entitled Study on the Biological Activity of Amomum tsao-ko Polysaccharides and Development of Functional Products (202505AP120026).

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 that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Figure 1. Chromatographic elution pattern of crude C. cassia polysaccharides using a DEAE-Sepharose Fast Flow column.
Figure 1. Chromatographic elution pattern of crude C. cassia polysaccharides using a DEAE-Sepharose Fast Flow column.
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Figure 2. The monosaccharide composition of polysaccharides from C. cassia using GC-MS analysis; (A) Crude, (B) F1 and (C) F2. The black line represents the total ion chromatogram, and the red line indicates the integration baseline.
Figure 2. The monosaccharide composition of polysaccharides from C. cassia using GC-MS analysis; (A) Crude, (B) F1 and (C) F2. The black line represents the total ion chromatogram, and the red line indicates the integration baseline.
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Figure 3. Refractive index (RI) chromatograms of polysaccharides from C. cassia: (A) crude polysaccharide, (B) Fraction F1, and (C) Fraction F2.
Figure 3. Refractive index (RI) chromatograms of polysaccharides from C. cassia: (A) crude polysaccharide, (B) Fraction F1, and (C) Fraction F2.
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Figure 4. Proliferation rate (A) and nitrite production (B) in RAW 264.7 macrophages after treatment with crude polysaccharide, F1, or F2. Symbols x–z denote statistically significant differences among samples (p < 0.05), whereas letters a–c represent significant differences among concentrations within the same sample (p < 0.05).
Figure 4. Proliferation rate (A) and nitrite production (B) in RAW 264.7 macrophages after treatment with crude polysaccharide, F1, or F2. Symbols x–z denote statistically significant differences among samples (p < 0.05), whereas letters a–c represent significant differences among concentrations within the same sample (p < 0.05).
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Figure 5. Impact of C. cassia polysaccharides on LPS-stimulated cytokine transcription and protein phosphorylation in RAW 264.7 macrophages. (A–E) Relative mRNA levels of iNOS, TNF-α, IL-1β, IL-6, and IL-10; (F) Phosphorylation status of p65, JNK, ERK, and p38. LPS (2 μg/mL) served as the positive control. Different lowercase letters indicate significant differences among treatment groups (p < 0.05).
Figure 5. Impact of C. cassia polysaccharides on LPS-stimulated cytokine transcription and protein phosphorylation in RAW 264.7 macrophages. (A–E) Relative mRNA levels of iNOS, TNF-α, IL-1β, IL-6, and IL-10; (F) Phosphorylation status of p65, JNK, ERK, and p38. LPS (2 μg/mL) served as the positive control. Different lowercase letters indicate significant differences among treatment groups (p < 0.05).
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Figure 6. Effects of C. cassia polysaccharides on RAW 264.7 cell surface marker expression analyzed by flow cytometry. (A) CD40 expression levels. (B) CD11b expression levels. In the histograms, the blue (A) and green (B) lines represent unstained control cells, while the red lines represent antibody-stained cells. The red brackets (P2) indicate the gated cell population used for quantitative analysis. Treatment groups labeled with different lowercase letters differ significantly from one another (p < 0.05).
Figure 6. Effects of C. cassia polysaccharides on RAW 264.7 cell surface marker expression analyzed by flow cytometry. (A) CD40 expression levels. (B) CD11b expression levels. In the histograms, the blue (A) and green (B) lines represent unstained control cells, while the red lines represent antibody-stained cells. The red brackets (P2) indicate the gated cell population used for quantitative analysis. Treatment groups labeled with different lowercase letters differ significantly from one another (p < 0.05).
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Table 1. Chemical composition of polysaccharides isolated from C. cassia.
Table 1. Chemical composition of polysaccharides isolated from C. cassia.
SamplesYield (%)Chemical Contents (%)
CarbohydrateProteinSulfateUronic Acid
Crude42.4 ± 1.449.3 ± 3.810.3 ± 0.511.3 ± 1.85.6 ± 0.3
F1 (DW)24.4 ± 2.354.8 ± 1.44.8 ± 0.28.1 ± 1.63.9 ± 0.1
F2 (0.5 mol/L NaCI)15.7 ± 1.261.1 ± 6.08.0 ± 0.19.5 ± 1.13.7 ± 0.7
Note: Crude yield, (weight of crude polysaccharide/weight of C. cassia) × 100; F1 and F2 yield, (weight of fractions/weight of crude injected into ion-exchange chromatography) × 100. Data are expressed as mean ± SD (n = 3).
Table 2. Monosaccharide composition of polysaccharides from C. cassia.
Table 2. Monosaccharide composition of polysaccharides from C. cassia.
Monosaccharide Content (%)Samples
CrudeF1F2
Rhamnose0.4 ± 0.00.1 ± 0.00.9 ± 0.1
Arabinose2.2 ± 0.00.8 ± 0.13.5 ± 0.8
Xylose0.8 ± 0.10.3 ± 0.01.0 ± 0.2
Glucose94.7 ± 1.198.8 ± 0.291.7 ± 0.5
Galactose1.2 ± 0.3ND2.8 ± 0.6
Note: Data are expressed as mean ± SD (n = 3). ND: not detected.
Table 3. Average molecular weight (Mw) and radius of gyration (Rg) of polysaccharides from C. cassia.
Table 3. Average molecular weight (Mw) and radius of gyration (Rg) of polysaccharides from C. cassia.
SamplesCrudeF1F2
MW (kDa)Peak I4321.3 ± 338.32919.1 ± 358.769.0 ± 3.3
Peak II214.1 ± 3.872.4 ± 1.546.1 ± 2.1
Rg (nm)Peak I60.2 ± 2.059.8 ± 1.570.4 ± 0.3
Peak II71.7 ± 1.871.2 ± 0.170.9 ± 0.4
SVg (cm3/g)Peak I0.13 ± 0.00.18 ± 0.012.8 ± 0.6
Peak II4.3 ± 0.312.6 ± 0.319.5 ± 0.9
Note: SVg = 4/3π(Rg × 108)3/(Mw/N) = 2.522 Rg3/Mw. Note: Data are expressed as mean ± SD (n = 3).
Table 4. Glycosidic linkages analysis of F2 polysaccharide isolated from C. cassia.
Table 4. Glycosidic linkages analysis of F2 polysaccharide isolated from C. cassia.
Retention
Time (min)		Methylation ProductGlycosidic LinkagePeak Area (%)
5.7281,4-di-O-acetyl-2,3,5-tri-O-methyl- arabinitolAra → 11.2 ± 0.2
7.1701,3,4-tri-O-acetyl-2,5-di-O-methyl-arabinitol1 → 3 Ara0.5 ± 0.1
7.6981,4,5-tri-O-acetyl-2,3-di-O-methyl- arabinitol1 →5 Ara0.9 ± 0.2
8.4181,5-di-O-acetyl-2,3,4,6-tretra-O-methyl- glucoseGlu → 14.1 ± 0.3
10.3221,4,5-tri-O-acetyl-2,3,6-tri-O- methyl- glucose1 → 4 Glu92.3 ± 0.1
10.5171,5,6-tri-O-acetyl-2,3,6-tri-O-methyl- glucose1 → 6 Glu0.1 ± 0.1
11.2801,3,4,5-tretra-O-acetyl-2,6-di-O-methyl- glucose1 → 3,4 Glu0.4 ± 0.1
11.5651,2,4,5-tetra-O-acetyl-1-deuterio-3,6-di-O-methyl-D-glucitol1 → 2,4 Glc0.5 ± 0.2
Note: Data are expressed as mean ± SD (n = 3).
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Dong, J.; Zhang, P.; Palanisamy, S.; Yin, H.; Zhang, Q.; Fang, C.; You, S.; Ge, Y. Purification, Structural Characterization, and Immunomodulatory Activity of Polysaccharides from Cinnamomum cassia. Foods 2026, 15, 927. https://doi.org/10.3390/foods15050927

AMA Style

Dong J, Zhang P, Palanisamy S, Yin H, Zhang Q, Fang C, You S, Ge Y. Purification, Structural Characterization, and Immunomodulatory Activity of Polysaccharides from Cinnamomum cassia. Foods. 2026; 15(5):927. https://doi.org/10.3390/foods15050927

Chicago/Turabian Style

Dong, Jinya, Peng Zhang, Subramanian Palanisamy, Huajie Yin, Qiyuan Zhang, Chongye Fang, SangGuan You, and Yunfei Ge. 2026. "Purification, Structural Characterization, and Immunomodulatory Activity of Polysaccharides from Cinnamomum cassia" Foods 15, no. 5: 927. https://doi.org/10.3390/foods15050927

APA Style

Dong, J., Zhang, P., Palanisamy, S., Yin, H., Zhang, Q., Fang, C., You, S., & Ge, Y. (2026). Purification, Structural Characterization, and Immunomodulatory Activity of Polysaccharides from Cinnamomum cassia. Foods, 15(5), 927. https://doi.org/10.3390/foods15050927

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