A Neutral Glucan Extracted from Dried Ginger (Zingiberis Rhizoma): Preparation, Structure Characterization, and Immunomodulatory Activity

• Abstract
• Introduction
• Results
• Discussion
• Materials and Methods
• Materials and chemicals
• Extraction and separation of polysaccharides
• Molecular weight determination and monosaccharide composition analysis
• FT-IR analysis
• Methylation and GC-MS analysis
• Nuclear magnetic resonance spectroscopy (NMR) analysis
• Cell line and cell culture
• RNA extraction and qPCR
• Intracellular reactive oxygen species (ROS) determination
• Western blotting analysis
• Statistical analysis
• Contributorsʼ Statement
• References

Abstract

A neutral glucan, GJ0D, was obtained from dried ginger (Zingiberis rhizoma) by enzymatic extraction and purification with column chromatography. The fine structure of GJ0D was assessed through monosaccharide composition analysis, methylation, and two-dimensional nuclear magnetic resonance. GJ0D has a relative molecular weight of 4.0 KDa and possesses a backbone consisting of 1,4-linked α-Glcp with substitution at C-6 of Glcp by T-Glcp. Immunoactivity assessment showed that GJ0D significantly upregulates the expression of IL-6, IL-1β, and TNF-α in RAW264.7 cells. The reactive oxygen species (ROS) production was also increased in RAW264.7 cells. In addition, the expression of several proteins associated with immune activation signaling pathways including TLR4, the phosphorylation of IKKβ, and NF-κB (p100 and p52) were significantly upregulated by GJ0D. These results suggest that GJ0D could promote inflammation through the TLR4/IKKβ/P100 signaling pathway, suggesting a potential application as an immunomodulating agent.

Introduction

Ginger, scientifically known as Zingiber officinale Rosc., is a perennial herbaceous plant belonging to the Zingiberaceae family, which is widely cultivated in Asia and has a long history of cultivation. During the past few decades, ginger has become one of the most extensively researched natural plant resources due to its rich content of bioactive constituents [1]. Ginger is widely used as both a medicinal resource and a versatile dual-use food. It is meanwhile also a common food-flavoring agent, a natural source of functional foods, and a key component of nutraceuticals. In Chinese medicine, ginger, dried ginger (Zingiberis rhizoma), and gunpowder ginger have been employed for various clinical purposes. They serve as common over-the-counter herbal remedies in China for addressing a range of ailments, including the common cold, coughs, and gastrointestinal disturbances [2]. Ginger contains various active compounds such as polysaccharides, polyphenols, terpenoids, and curcumin and its derivatives [3]. A wide range of bioactivities of ginger have been reported including anti-inflammatory [4], cytotoxic [5], immunomodulatory [6], blood-glucose-decreasing [7], and antioxidant activities [8]. Research on various forms of ginger has revealed differences in the composition and bioactivity between dried ginger and fresh ginger. For example, 6-gingerol, found in fresh ginger, undergoes transformation into 6-shogaol during the drying process. Studies have shown that 6-shogaol demonstrates enhanced anti-cancer, antioxidant, and anti-inflammatory properties in comparison to 6-gingerol [9], [10], [11]. Additionally, dried ginger has been observed to possess enhanced antioxidant properties [12].

Ginger polysaccharides, being one of gingerʼs active components, have garnered much interest. Previous research has shown that ginger polysaccharides inhibit the expression of inflammatory cytokines in mice treated with dextran sodium sulfate (DSS), thereby regulating gastrointestinal immunomodulation and repairing the intestinal barrier, consequently improving the intestinal flora environment [13]. Furthermore, it is noted that various extraction methods yield differing results regarding the structure and pharmacological activities of ginger polysaccharides. Chen et al. [8] compared the effects of different extraction methods on the yield, chemical composition, structural characterization, and hypoglycemic and antioxidant activities of ginger stem and leaf polysaccharides (GSLP). Their findings revealed that polysaccharides obtained by enzyme-assisted extraction had the highest extraction yield compared to those prepared by hot-water extraction, ultrasound-assisted extraction, and alkaline-solution extraction. Wang et al. [14] used an enzymatic method to obtain a ginger polysaccharide with cytotoxic properties. Currently, few studies have been reported on the immunomodulatory activity of dried ginger polysaccharides. In organisms, appropriate inflammatory and immune responses are crucial for pathogen recognition, defense against pathogen invasion, and the removal of internal environmental risk factors. However, if inflammation persists unchecked, it can contribute to the development of autoimmune or autoinflammatory diseases, neurodegenerative disorders, and cancer [15], [16]. Therefore, further investigation into the precise chemical structure of ginger polysaccharides, as well as a comprehensive assessment of their immunomodulatory activity and underlying mechanisms, is essential.

Here, we analyzed the structural features of GJ0D, a dried ginger polysaccharide extracted through a combination of enzymatic extraction and column chromatography. Preliminary pharmacological investigations indicate that GJ0D exhibits immunostimulatory activity. GJ0D could upregulate the expression of IL-6, IL-1β and TNF-α in RAW264.7 cells and significantly enhance ROS production. In addition, GJ0D enhanced the expression of TLR4, the phosphorylation of IKKβ, and the expression of downstream NF-κB. Based on these results, we hypothesize that the neutral glucan GJ0D from dried ginger may promote inflammation partly through the TLR4/IKKβ/P100 signaling pathway. This study provides a scientific basis for developing dried ginger polysaccharide GJ0D as a potential immunomodulator.

Results

The extraction flow chart of polysaccharides from dried ginger is shown in [Fig. 1] The main fraction named as GJ0D was obtained by purification with a DEAE Sepharose column followed by a Sephacryl S-100 HR column ([Fig. 2 a] and [b]) in 5.62% total yield. A distinct symmetrical peak was observed in the HPGPC chromatogram, which indicated that GJ0D was a homogeneous polysaccharide with a relative molecular weight of 4.0 KDa ([Fig. 2 c]). The monosaccharide composition of GJ0D is shown in [Fig. 2 d]. Glucose was the major monosaccharide, which suggested that GJ0D was a neutral glucan.

Fourier transform infrared (FT-IR) analysis is commonly used in the determination of the molecular structure and chemical composition of polysaccharides. The FT-IR spectrum of GJ0D ([Fig. 3]) exhibited characteristic absorption peaks within the range of 4000 to 400 cm⁻¹. Specifically, the absorption peaks observed at 3293 cm⁻¹ and 2926 cm⁻¹ were associated with the stretching vibration of O-H and C – H bonds, respectively [17]. Furthermore, the absorption peaks detected at 1658 cm⁻¹ and 1336 cm⁻¹ were attributed to the asymmetric stretching vibration of C=O bonds and the symmetric stretching vibration, respectively. The absorption peak around 1146 cm⁻¹ was due to the stretching vibration of C – O on the pyranose ring. Finally, the absorption peaks at 929 cm⁻¹ and 839 cm⁻¹ are indicative of glucopyranose derivatives, suggesting that this polysaccharide consists of pyranose rings linked by α-glycosidic bonds.

To determine the glycosidic bond type, GJ0D was permethylated, hydrolyzed, and then analyzed for its partially methylated ethylene glycol acetates (PMAA) by GC-MS. The total ion chromatogram and glycosidic bond type analysis results of GJ0D are shown in [Fig. 4] and [Table 1]. GJ0D was found to contain three major glycosidic units consisting of T-Glcp (22.3%), 1,4-conjugated Glcp (47.5%), and 1,4,6-conjugated Glcp (18.4%). A small amount of 1,6-conjugated Glcp (7.9%) and 1,3,4-conjugated Glcp (3.9%) residues were also present. These findings suggest that the core structure of GJ0D is primarily composed of 1,4-linked α-Glcp with T-Glcp substituting at the C-6 position, representing a predominantly branched glycan. These structural elements aligned with the results from the monosaccharide analysis.

In order to characterize the fine structure of GJ0D, 1D-NMR and 2D-NMR spectra were recorded and the proton and carbon chemical shifts of each type of residue were assigned, with the main glycosidic linkages identified as A – E ([Table 2]). The ¹H and ¹³C NMR spectra of GJ0D are shown in [Fig. 5] The ¹H and ¹³C signals ([Table 2]) were assigned through ¹H-¹H-COSY ([Fig. 5 c]), HSQC (FIG.[5 d]), and HMBC ([Fig. 5 e]) experiments, in combination with methylation findings and literature data [6], [18], [19]. The ¹³C-NMR spectrum showed five major anomeric carbon signals (99.8, 101.2, 100.8, 101.4, and 101.2 ppm), which were assigned to C-1 of T-α-Glcp, 1,4-linked-α-Glcp, 1,6-linked-α-Glcp, 1,3,4-linked-α-Glcp, and 1,4,6-linked-α-Glcp, respectively. The corresponding anomeric proton signals were assigned from the HSQC spectrum. The overlapping peaks around 5.40 ppm belong to H-1 of 1,4-linked-α-Glcp, 1,6-linked-α-Glcp, 1,3,4-linked-α-Glcp, and 1,4,6-linked-α-Glcp, respectively. The signal at 5.04 ppm was assigned to H-1 of T-α-Glcp. Based on 2D NMR spectra analysis, all other ¹H and ¹³C resonances were identified as listed in [Table 2]. The signals at 78.5 ppm, 79.3 ppm, and 79.3 ppm belonged to C-4 of 1,4-linked-α-Glcp, 1,3,4-linked-α-Glcp, and 1,4,6-linked-α-Glcp, respectively, while the peaks at 68.3 ppm and 68.6 ppm were due to C-6 of 1,6-linked-α-Glcp and 1,4,6-linked-α-Glcp. In the HMBC spectrum, the presence of cross peaks between the proton H-1 of residue A (5.04 ppm) and the carbon C-6 of residue E (68.6 ppm) suggested that the terminal residue α-D-Glcp was attached to the 1,4,6-linked-α-Glcp at C-6. Further correlations between H-1/residue B (5.43 ppm) and C-4/residue E (79.3 ppm), as well as H-1/residue E (5.43 ppm) and C-4/residue B (78.5 ppm), were observed in the HMBC spectrum ([Fig. 2 e]), which indicated that the main sugar linkage type of GJ0D was → 4)-α-D-Glcp →. The above data in conjunction with the methylation results reveal that the molar ratio of T-Glcp (22.3%), 1,4-linked Glcp (47.5%), and 1,4,6-linked Glcp (18.4%) approximates a ratio of 2 : 5 : 2. The analysis further indicates that GJ0D possesses a 1,4-linked-D-Glcp backbone, with a terminal residue of α-D-Glcp-(1 → attached to the → 4,6)-α-D-Glcp-(1 → at the C-6 position. [Fig. 5 f] shows the repeating unit for GJ0D based on this analysis.

ROS is a collective term for aggressive oxidative species including superoxide anion, hydroxyl radical, and hydrogen peroxide, which are mainly derived from the mitochondrial respiratory chain. It is established that optimal levels of reactive oxygen species (ROS) are essential for maintaining cellular homeostasis, whereas excessive ROS production can induce apoptosis, cellular injury, and death. Therefore, the release of ROS serves as a crucial indicator of activated macrophages. As shown in [Fig. 6], ROS production in the blank group was much lower than those in the LPS group (1 µg/mL) and the GJ0D-treated group. In addition, GJ0D increased ROS production in a significant dose-dependent manner. These results demonstrate that GJ0D has a significant effect on RAW264.7 cells, especially in increasing the ROS production.

In order to address whether GJ0D could promote inflammation, we first employed the classic LPS-induced RAW264.7 model. As shown in [Fig. 7], LPS stimulation was found to promote the mRNA expression of IL-6, IL-1β, and TNF-α. As we expected, the combination treatment of GJ0D with LPS sharply enhanced this expression promotion. Subsequently, we treated the cells with LPS or GL0D alone and then detected the mRNA expression of the aforementioned genes. Similarly, GJ0D could upregulate the expression of IL-6, IL-1β, and TNF-α at the mRNA level even better than the LPS group, further suggesting that GJ0D exhibits a promising inflammation-promoting effect.

Subsequently, we investigated the potential mechanism by which GJ0D may promote inflammation. The NF-κB signaling pathway is widely recognized for its pivotal role in inflammation. Thus, we conducted a Western blot analysis to evaluate the effect of GJ0D on this specific signaling pathway. The results depicted in [Fig. 7] show a significant increase in the phosphorylation of IKKβ following treatment with GJ0D at concentrations of 500 and 1000 µg/mL. Additionally, the expression of the downstream NF-κB (p100 and p52) was also upregulated significantly. Many literature sources have emphasized the importance of a toll-like receptor in inflammation [20]. Interestingly, GJ0D could enhance the TLR4 expression. Based on the above results, we speculate that GJ0D could promote inflammation partially via the TLR4/IKKβ/P100 signaling pathway. Nevertheless, the precise mechanism still needs further exploration.

Discussion

Polysaccharides are an important group of constituents in plants, which do not only play a role as cytoskeletal support and energy supply. Numerous studies have revealed the diverse biological activities and low toxic effects of polysaccharides [21], [22], [23], [24]. Moreover, as to the physical properties, polysaccharides from plants exhibit notable rheological properties, including thickening, stabilizing, gelling, and emulsifying capabilities [25].

The biological activity of polysaccharides generally depends on their structural characteristics. Different extraction and preparation methods lead to different structures of polysaccharides, even for polysaccharides of the same plant origin [26], [27]. The monosaccharide composition, glycosidic linkage types, and spatial configuration of polysaccharides are important factors contributing to the structural diversity. Therefore, the structural characterization of polysaccharides is essential in view of their potential use as a bioactive product.

In this study, we have characterized the structure of the neutral polysaccharide fraction GJ0D from dried ginger that was obtained by combined enzymatic extraction and column isolation. GJ0D was found to have a low molecular weight of 4.0 KDa and was identified as an α-glucan with a 1,4-linkeded α-D-Glcp backbone. Usually, starch constitutes the main polysaccharide in plant tubers. The presence of starch affects the purification and activity of other structural polysaccharides in plants. In this study, starch was initially removed by amylase. Interestingly, GJ0D was still an α-glucan but a non-starchy glucan.

Up to now, several ginger polysaccharides with different structural characteristics have been obtained through different extraction methods. An acidic heteropolysaccharide with a molecular weight of 100.17 KDa was obtained from ginger through ultrasound extraction [28]. Various polysaccharidic components were isolated from dried ginger through the use of distinct extraction techniques including hot-water extraction, enzyme-assisted extraction, and ultrasonic cell-grinder extraction. The molecular weights varied among them, ranging from 11.81 to 1831.75 KDa [5]. Compared with previous studies, GJ0D has a low molecular weight, partly due to the combined enzymatic extraction and amylase degradation. According to literature sources, the variance in molecular weight plays a crucial role for the variability in the immunological activity of polysaccharides [29]. Zhou et al. [30] extracted multiple polysaccharides from Porphyridium cruentum of various molecular sizes and discovered that those with lower molecular weights exhibited superior immunoenhancing activity. Here, RAW264.7 cells were used as a model to assess the immunomodulatory activity. GJ0D was able to up-regulate the expression of IL-6, IL-1β, and TNF-α at the mRNA level even better than the LPS group, suggesting that GJ0D had a favorable inflammatory effect. Stimulation of macrophages is recognized as a key way in which polysaccharides exert immune effects. Multiple pathways typically play a role in this process, such as enhancing the growth of macrophages, boosting phagocytic function, raising levels of NO and ROS, and regulating the secretion of cytokines and chemokines [31]. In the present study, we showed that GJ0D at different concentrations could significantly stimulate macrophage proliferation and ROS production, suggesting that GJ0D can enhance immune response through macrophage stimulation. Immunity is a function of inflammation, and inflammation plays a major role in eliminating microbes.

Toll-like receptors (TLRs) recognize various molecules associated with pathogens, with their main role being the regulation of inflammatory and immune reactions. Our work revealed that GJ0D lead to increased expression of the TLR4 gene in RAW264.7 cells. However, upregulating gene expression does not necessarily imply that GJ0D directly binds to TLR4 to activate it. It is important to differentiate between the stimulation of gene expression and the direct activation of receptor signaling pathways. The former may increase the number of receptors available for signaling, while the latter involves ligand–receptor interactions that trigger downstream signaling cascades [32].

GJ0D could potentially stimulate TLR4 expression indirectly, possibly through the activation of other signaling molecules or pathways that subsequently enhance TLR4 transcription. For example, GJ0D may activate pathways leading to the production of other cytokines or signaling molecules that, in turn, stimulate TLR4 expression. To ascertain whether GJ0D directly binds to TLR4, further studies are needed. Techniques such as surface plasmon resonance (SPR) or co-immunoprecipitation could help establish whether GJ0D interacts directly with TLR4.

In summary, while GJ0D has been shown to upregulate TLR4 expression, the mechanism by which it influences TLR4 signaling remains to be fully elucidated. Addressing this gap in knowledge will enhance the understanding of GJ0D’s immunomodulatory properties and its potential applications in clinical settings. Further research is essential to clarify whether GJ0D functions as a direct ligand for TLR4 or acts through indirect pathways to modulate immune responses.

Our findings suggest that GJ0D could serve as a potential immune-modulating agent. By promoting inflammation through the activation of the TLR4/IKKβ/NF-κB signaling pathway, GJ0D may enhance the bodyʼs immune responses, which could be beneficial in various contexts, such as infectious diseases or immune deficiencies. Further research, including in vivo studies and clinical trials, would be necessary to fully elucidate its effects and potential applications in immune modulation.

Materials and Methods

Materials and chemicals

The dried ginger (Zingiberis rhizoma) used in this study was obtained from Suzhou TCM Hospital, Jiangsu, China and authenticated by Prof. Lurong Zhang of the Suzhou Academy of Wumen Chinese Medicine, Suzhou TCM Hospital Affiliated to Nanjing University of Chinese Medicine, Suzhou, Jiangsu, China. A voucher specimen (No. 202 401) was deposited in Suzhou TCM Hospital Affiliated to Nanjing University of Chinese Medicine, Suzhou, Jiangsu, China. RAW264.7 cells were obtained from the Chinese Academy of Sciences in Shanghai, China. The reactive oxygen assay kit was purchased from Beyotime Biotechnology Co., Ltd. Trypsin (1800 U/mg) was provided by Dulai Biotechnology Co., Ltd., and papain (25 U/mg) was obtained from Yuanye Biotechnology Co., Ltd. Monosaccharide standards were obtained from Fluka, while trifluoroacetic acid (TFA, purity 99%) and 1-phenyl-3-methyl-5-pyrazolone (PMP, purity 99%) were purchased from Guoyao Co., Ltd. All other reagents used were of analytical grade.

Extraction and separation of polysaccharides

The dried ginger powder (200 g) was soaked in acetone (300 mL) and replaced every 12 h at r. t. (total time 36 h) for defatting. The mixture was centrifuged (4000 rcf for 20 min), and the solid fraction was collected. After drying, 165.6 g of dried defatted ginger powder was obtained, which was suspended in distilled water at a ratio of 1 : 30 and treated with 2.5% papain by mass. The extraction was conducted at pH 6 and 60 °C for 12 h, followed by inactivation at 100 °C for 15 min. After cooling, the pH was adjusted back to 8, and 2.5% trypsin was added. The mixture was stirred at 37 °C for 12 h, followed by inactivation at 100 °C for 15 min. After cooling, the mixture was centrifuged, and the supernatant was concentrated and dialyzed using a membrane with a cut-off of 8.0 KDa for 2 days. The resulting solution was then freeze-dried to obtain the dried ginger crude polysaccharide fraction GJ (13.1 g, 7.93%).

Starch iodine test paper revealed the presence of straight chain starch in GJ. Prior to further separation, α-amylase was added to the aqueous solution of GJ (200 mg) at a ratio of 25 : 1 (w/w) sample/amylase. Enzymatic digestion was carried out with stirring at 57 °C for 6 h [33]. After confirming the absence of starch residue, dialysis and lyophilization afforded GJD (84 mg, 42%). Starch-removed polysaccharides GJD was purified on a DEAE-52 cellulose column (Cl⁻, 120 cm × 6 cm). The elution was performed in a gradient manner using distilled water: 0.1 M NaCl, 0.2 M NaCl, and finally 0.3 M NaCl. A steady flow pump was used to maintain a flow rate of 1.0 mL/min, and 6 mL of eluate was collected in each tube. Following elution with distilled water, the first portion was combined, concentrated, and freeze-dried to yield crude polysaccharide GJ0D (165 mg). Final purification using a S-100 high-resolution column, eluted with 0.2 M NaCl at a flow rate of 0.3 mL/min, afforded homogeneous GJ0D (106 mg, 64.2% yield).

Molecular weight determination and monosaccharide composition analysis

The purity and molecular weight of GJ0D were determined by the HPGPC method [34]. The chromatographic columns were TOSOH TSK PWXL G4000 and TSK PWXL G2500 in tandem, the mobile phase was 0.15 mol/L NaNO₃ at a flow rate of 0.5 mL/min, and the column temperature was 35 °C. A calibration curve was constructed based on different molecular weights of T series dextran standards. The data were analyzed with Empower 3 (Waters) software. The monosaccharide composition of GJ0D was analyzed by a PMP-HPLC precolumn derivatization method [34]. Briefly, GJ0D (2 mg) was hydrolyzed in 2 M TFA at 110℃ for 4 h. Methanol was then added and evaporated repeatedly to remove TFA. The hydrolyzed monosaccharides were mixed with a proper amount of PMP at 70℃ for 1 h, and excess PMP was then removed by chloroform extraction. The derivatized monosaccharides were analyzed by HPLC with UV detection at 250 nm.

FT-IR analysis

Dried GJ0D (1 mg) was analyzed with a Spotlight 400 FTIR spectrometer (PerkinElmer) in the range of 4000 to 400 cm⁻¹. The spectra were processed using Omnic 9.2 (Thermo Scientific) and Origin 2018 (Origin Lab) software.

Methylation and GC-MS analysis

Then, 20 mg of vacuum-dried GJ0D was dissolved in dimethyl sulfoxide, and anhydrous sodium hydroxide powder was added and stirred until complete dissolution. The mixture from the methylation reaction was then added dropwise with iodomethane on an ice bath and stirred at room temperature for 30 min before adding distilled water until the reaction was terminated. The reaction solution was dialyzed against distilled water three times. The samples were freeze-dried to obtain methylated polysaccharides. The hydroxyl groups of polysaccharides were detected by IR to determine whether they were completely methylated or not, and if incomplete methylation was observed, the aforementioned procedure was repeated until complete methylation [35]. Finally, partially methylated alditol acetates (PMAAs) were prepared and analyzed using gas chromatography–mass spectrometry (GC-MS).

Nuclear magnetic resonance spectroscopy (NMR) analysis

GJ0D (30 mg) was suspended in 0.5 mL of D₂O (99% atom, Aldrich). One-dimensional and two-dimensional NMR spectra were recorded on a Varian Mercury 600 MHz NMR spectrometer, equipped with a helium-cooled cryoprobe. ¹H and ¹³C NMR chemical shifts are reported in ppm with the acetone solvent signal as internal reference. Then, 2D-NMR (1H-1H-COSY, HSQC, and HMBC) were recorded for signal assignment. All data were processed using MestReNova14.2.1 (Mestrelab Research) software.

Cell line and cell culture

RAW264.7 cells were cultured in Dulbeccoʼs modified Eagle medium (DMEM) from Corning, supplemented with 15% fetal bovine serum (FBS) from Gibco and 1% penicillin/streptomycin from Meilunbio. The cells were cultured at 37 °C in a 5% CO₂ atmosphere.

RNA extraction and qPCR

RAW264.7 cells were treated with lipopolysaccharide (LPS) at a concentration of 1 µg/mL (Sigma-Aldrich) or GJ0D at various concentrations for a 24-hour period. Total RNA was extracted using the EZ-press RNA purification kit (EZBioscience) and reverse transcribed into cDNA using the PrimeScriptTM 1st Strand cDNA synthesis kit (Takara) following the manufacturerʼs instructions. Quantitative PCR (qPCR) was conducted using a V7 PCR system (Applied Biosystems) and the SYBR Green Premix Ex Taq kit (Takara). Two replicates were analyzed for each sample to determine the Ct value of the target transcripts. The relative mRNA expression was defined as 2-[(Ct of gene) – (Ct of GAPDH)]. The primer sequences used are listed in [Table 3].

Intracellular reactive oxygen species (ROS) determination

RAW264.7 cells were seeded into 96-well plates (3 × 10⁴ cells/well) and allowed to adhere overnight. Then, the medium was replaced with medium with the GJ0D sample at different concentrations of 0, 25, 50, 100, 200, 400, 800, and 1000 µg/mL, respectively, and the cells were cultured for 24 h. Four replicates were set up in each group. The amount of ROS present within cells was measured by staining them with dichlorofluorescin diacetate (DCFH-DA, Beyotime) for 30 min, then washing the cells twice with PBS and analyzing them with a fluorescent microscope. LPS (1 µg/mL) was used as positive control.

Western blotting analysis

RAW264.7 cells were incubated with LPS or GJ0D at different concentrations in 6-well plates for 24 h. Then, the total protein was extracted by RIPA lysate (Thermo Scientific) containing protease inhibitors (Selleck). The BCA assay kit (Takara) was used to determine the protein content in each group. Following separation on SDS-PAGE gels, the protein samples were transferred to PVDF membranes for examination. Primary and secondary antibodies were sequentially incubated with the membrane, and the density of the resultant bands was quantified using KwikQuant Image Analyzer software. The primary antibodies used were IKKβ antibody (1 : 1500, Cat No. CY5636, Abways), Phospho-IKKα/β – S176/180 antibody (1 : 1000, Cat No. AP0546, Abclonal), TLR4 antibody (1 : 1000, Cat No. SC-10741, Santa Cruz), and NF-κB p100/p52 antibody (1 : 1000, Cat No. 4882, Cell Signaling Technology).

Statistical analysis

The data were expressed as the mean ± standard deviation derived from a minimum of three replicates. OriginPro 2018 (OriginLab), GraphPad Prism 8.0 (GraphPad Software), and SPSS 27 (IBM) were applied for data processing. Group comparisons were conducted using Studentʼs t-test, one-way ANOVA, or two-way ANOVA. Statistical significance was defined as p-value < 0.05.

Contributorsʼ Statement

Conception and design of the work: Peipei Wang and Jingdong Gao;data collection: Long Sun, Xing Ni, and Yulin Liu;analysis and interpretation of the data: Long Sun, Xing Ni, and Yulin Liu;statistical analysis: Yantao Jiang;drafting the manuscript: Long Sun, Xing Ni, Yulin Liu,Peipei Wang and Jingdong Gao;critical revision of the manuscript: Peipei Wang and Jingdong Gao.

Conflict of Interest

The authors declare that they have no conflict of interest.

• References

• 1 Srinivasan K. Ginger rhizomes (Zingiber officinale): A spice with multiple health beneficial potentials. PharmaNutrition 2017; 5: 18-28
  PubMedSearch in Google Scholar
• 2 Gupta S, Sharma A. Medicinal properties of Zingiber officinale Roscoe – A Review. IOSR-JPBS 2014; 9: 124-129
  PubMedSearch in Google Scholar
• 3 Mao QQ, Xu XY, Cao SY, Gan RY, Corke H, Beta T, Li HB. Bioactive compounds and bioactivities of ginger (Zingiber officinale Roscoe). Foods 2019; 8: 185
  CrossrefPubMedSearch in Google Scholar
• 4 Ballester P, Cerdá B, Arcusa R, Marhuenda J, Yamedjeu K, Zafrilla P. Effect of ginger on inflammatory diseases. Molecules 2022; 27: 7223
  PubMedSearch in Google Scholar
• 5 Liao DW, Cheng C, Liu JP, Zhao LY, Huang DC, Chen GT. Characterization and antitumor activities of polysaccharides obtained from ginger (Zingiber officinale) by different extraction methods. Int J Biol Macromol 2020; 152: 894-903
  PubMedSearch in Google Scholar
• 6 Yang X, Wei S, Lu X, Qiao X, Simal-Gandara J, Capanoglu E, Woźniak Ł, Zou L, Cao H, Xiao J, Tang X, Li N. A neutral polysaccharide with a triple helix structure from ginger: Characterization and immunomodulatory activity. Food Chem 2021; 350: 129261
  PubMedSearch in Google Scholar
• 7 Chen X, Wang Z, Kan J. Polysaccharides from ginger stems and leaves: Effects of dual and triple frequency ultrasound assisted extraction on structural characteristics and biological activities. Food Biosci 2021; 42: 101166
  PubMedSearch in Google Scholar
• 8 Chen X, Chen G, Wang Z, Kan J. A comparison of a polysaccharide extracted from ginger (Zingiber officinale) stems and leaves using different methods: preparation, structure characteristics, and biological activities. Int J Biol Macromol 2020; 151: 635-649
  PubMedSearch in Google Scholar
• 9 Nonaka K, Bando M, Sakamoto E, Inagaki Y, Naruishi K, Yumoto H, Kido JI. 6-Shogaol inhibits advanced glycation end-products-induced IL-6 and ICAM-1 expression by regulating oxidative responses in human gingival fibroblasts. Molecules 2019; 24: 3705
  PubMedSearch in Google Scholar
• 10 Prasad S, Tyagi AK. Ginger and its constituents: Role in prevention and treatment of gastrointestinal cancer. Gastroenterol Res Pract 2015; 2015: 142979
  PubMedSearch in Google Scholar
• 11 Suekawa M, Ishige A, Yuasa K, Sudo K, Aburada M, Hosoya E. Pharmacological studies on ginger. I. Pharmacological actions of pungent constituents, (6)-gingerol and (6)-shogaol. J Pharmacobiodyn 1984; 7: 836-848
  PubMedSearch in Google Scholar
• 12 Gümüşay ÖA, Borazan AA, Ercal N, Demirkol O. Drying effects on the antioxidant properties of tomatoes and ginger. Food Chem 2015; 173: 156-162
  PubMedSearch in Google Scholar
• 13 Hao W, Chen Z, Yuan Q, Ma M, Gao C, Zhou Y, Zhou H, Wu X, Wu D, Farag MA, Wang S, Wang Y. Ginger polysaccharides relieve ulcerative colitis via maintaining intestinal barrier integrity and gut microbiota modulation. Int J Biol Macromol 2022; 219: 730-739
  PubMedSearch in Google Scholar
• 14 Wang Y, Wang S, Song R, Cai J, Xu J, Tang X, Li N. Ginger polysaccharides induced cell cycle arrest and apoptosis in human hepatocellular carcinoma HepG2 cells. Int J Biol Macromol 2019; 123: 81-90
  PubMedSearch in Google Scholar
• 15 Aggarwal BB. Nuclear factor-κB: The enemy within. Cancer Cell 2004; 6: 203-208
  PubMedSearch in Google Scholar
• 16 Mantovani A. Inflammation by remote control. Nature 2005; 435: 752-753
  PubMedSearch in Google Scholar
• 17 Cao J, Tang D, Wang Y, Li X, Hong L, Sun C. Characteristics and immune-enhancing activity of pectic polysaccharides from sweet cherry (Prunus avium). Food Chem 2018; 254: 47-54
  PubMedSearch in Google Scholar
• 18 Wang P, Liao W, Fang J, Liu Q, Yao J, Hu M, Ding K. A glucan isolated from flowers of Lonicera japonica Thunb. inhibits aggregation and neurotoxicity of Aβ42. Carbohydr Polym 2014; 110: 142-147
  PubMedSearch in Google Scholar
• 19 Zhang S, Zhang C, Li M, Chen X, Ding K. Structural elucidation of a glucan from Crataegus pinnatifida and its bioactivity on intestinal bacteria strains. Int J Biol Macromol 2019; 128: 435-443
  PubMedSearch in Google Scholar
• 20 Lam JH, Baumgarth N. Toll-like receptor mediated inflammation directs B cells towards protective antiviral extrafollicular responses. Nat Commun 2023; 14: 3979
  PubMedSearch in Google Scholar
• 21 Benalaya I, Alves G, Lopes J, Silva LR. A review of natural polysaccharides: Sources, characteristics, properties, food, and pharmaceutical applications. Int J Mol Sci 2024; 25: 1322
  PubMedSearch in Google Scholar
• 22 Ji X, Cheng Y, Tian J, Zhang S, Jing Y, Shi M. Structural characterization of polysaccharide from jujube (Ziziphus jujuba Mill.) fruit. Chem Biol Technol Agric 2021; 8: 54
  PubMedSearch in Google Scholar
• 23 Ji X, Guo J, Ding D, Gao J, Hao L, Guo X, Liu Y. Structural characterization and antioxidant activity of a novel high-molecular-weight polysaccharide from Ziziphus Jujuba cv. Muzao. J Food Meas Charact 2022; 16: 2191-2200
  PubMedSearch in Google Scholar
• 24 ShanChen. Khan BM, Cheong KL, Liu Y. Pumpkin polysaccharides: Purification, characterization and hypoglycemic potential. Int J Biol Macromol 2019; 139: 842-849
  PubMedSearch in Google Scholar
• 25 Yang X, Li A, Li X, Sun L, Guo Y. An overview of classifications, properties of food polysaccharides and their links to applications in improving food textures. Trends Food Sci Tech 2020; 102: 1-15
  PubMedSearch in Google Scholar
• 26 Li Y, Zhang X, Li Y, Yang P, Zhang Z, Wu H, Zhu L, Liu Y. Preparation methods, structural characteristics, and biological activity of polysaccharides from Salvia miltiorrhiza: A review. J Ethnopharmacol 2023; 305: 116090
  PubMedSearch in Google Scholar
• 27 Wang Y, Zhang H. Advances in the extraction, purification, structural-property relationships and bioactive molecular mechanism of Flammulina velutipes polysaccharides: A review. Int J Biol Macromol 2021; 167: 528-538
  PubMedSearch in Google Scholar
• 28 Lin X, Xiao B, Liu J, Cao M, Yang Z, Zhao L, Chen G. An acidic heteropolysaccharide rich in galactose and arabinose derived from ginger: Structure and dynamics. Food Biosci 2023; 56: 103127
  PubMedSearch in Google Scholar
• 29 Li Z, Du Z, Wang Y, Feng Y, Zhang R, Yan X. Chemical Modification, Characterization, and Activity Changes of Land Plant Polysaccharides: A Review. Polymers (Basel) 2022; 14: 4161
  PubMedSearch in Google Scholar
• 30 Sun L, Wang L, Zhou Y. Immunomodulation and antitumor activities of different-molecular-weight polysaccharides from Porphyridium cruentum . Carbohydr Polym 2012; 87: 1206-1210
  PubMedSearch in Google Scholar
• 31 Yin M, Zhang Y, Li H. Advances in research on immunoregulation of macrophages by plant polysaccharides. Front Immunol 2019; 10: 145
  PubMedSearch in Google Scholar
• 32 Coutinho-Wolino KS, Almeida PP, Mafra D, Stockler-Pinto MB. Bioactive compounds modulating Toll-like 4 receptor (TLR4)-mediated inflammation: pathways involved and future perspectives. Nutr Res 2022; 107: 96-116
  PubMedSearch in Google Scholar
• 33 Ji M, Sun L, Zhang M, Liu Y, Zhang Z, Wang P. RN0D, a galactoglucan from Panax notoginseng flower induces cancer cell death via PINK1/Parkin mitophagy. Carbohydr Polym 2024; 332: 121889
  PubMedSearch in Google Scholar
• 34 Wang P, Zhang L, Yao J, Shi Y, Li P, Ding K. An arabinogalactan from flowers of Panax notoginseng inhibits angiogenesis by BMP2/Smad/Id1 signaling. Carbohydr Polym 2015; 121: 328-335
  PubMedSearch in Google Scholar
• 35 Cong Q, Chen H, Liao W, Xiao F, Wang P, Qin Y, Dong Q, Ding K. Structural characterization and effect on anti-angiogenic activity of a fucoidan from Sargassum fusiforme . Carbohydr Polym 2016; 136: 899-907
  PubMedSearch in Google Scholar

Correspondence

Publication History

Received: 11 November 2024

Accepted: 20 March 2025

Article published online:
14 April 2025

© 2025. Thieme. All rights reserved.

Georg Thieme Verlag KG
Oswald-Hesse-Straße 50, 70469 Stuttgart, Germany

• References

• 1 Srinivasan K. Ginger rhizomes (Zingiber officinale): A spice with multiple health beneficial potentials. PharmaNutrition 2017; 5: 18-28
  PubMedSearch in Google Scholar
• 2 Gupta S, Sharma A. Medicinal properties of Zingiber officinale Roscoe – A Review. IOSR-JPBS 2014; 9: 124-129
  PubMedSearch in Google Scholar
• 3 Mao QQ, Xu XY, Cao SY, Gan RY, Corke H, Beta T, Li HB. Bioactive compounds and bioactivities of ginger (Zingiber officinale Roscoe). Foods 2019; 8: 185
  CrossrefPubMedSearch in Google Scholar
• 4 Ballester P, Cerdá B, Arcusa R, Marhuenda J, Yamedjeu K, Zafrilla P. Effect of ginger on inflammatory diseases. Molecules 2022; 27: 7223
  PubMedSearch in Google Scholar
• 5 Liao DW, Cheng C, Liu JP, Zhao LY, Huang DC, Chen GT. Characterization and antitumor activities of polysaccharides obtained from ginger (Zingiber officinale) by different extraction methods. Int J Biol Macromol 2020; 152: 894-903
  PubMedSearch in Google Scholar
• 6 Yang X, Wei S, Lu X, Qiao X, Simal-Gandara J, Capanoglu E, Woźniak Ł, Zou L, Cao H, Xiao J, Tang X, Li N. A neutral polysaccharide with a triple helix structure from ginger: Characterization and immunomodulatory activity. Food Chem 2021; 350: 129261
  PubMedSearch in Google Scholar
• 7 Chen X, Wang Z, Kan J. Polysaccharides from ginger stems and leaves: Effects of dual and triple frequency ultrasound assisted extraction on structural characteristics and biological activities. Food Biosci 2021; 42: 101166
  PubMedSearch in Google Scholar
• 8 Chen X, Chen G, Wang Z, Kan J. A comparison of a polysaccharide extracted from ginger (Zingiber officinale) stems and leaves using different methods: preparation, structure characteristics, and biological activities. Int J Biol Macromol 2020; 151: 635-649
  PubMedSearch in Google Scholar
• 9 Nonaka K, Bando M, Sakamoto E, Inagaki Y, Naruishi K, Yumoto H, Kido JI. 6-Shogaol inhibits advanced glycation end-products-induced IL-6 and ICAM-1 expression by regulating oxidative responses in human gingival fibroblasts. Molecules 2019; 24: 3705
  PubMedSearch in Google Scholar
• 10 Prasad S, Tyagi AK. Ginger and its constituents: Role in prevention and treatment of gastrointestinal cancer. Gastroenterol Res Pract 2015; 2015: 142979
  PubMedSearch in Google Scholar
• 11 Suekawa M, Ishige A, Yuasa K, Sudo K, Aburada M, Hosoya E. Pharmacological studies on ginger. I. Pharmacological actions of pungent constituents, (6)-gingerol and (6)-shogaol. J Pharmacobiodyn 1984; 7: 836-848
  PubMedSearch in Google Scholar
• 12 Gümüşay ÖA, Borazan AA, Ercal N, Demirkol O. Drying effects on the antioxidant properties of tomatoes and ginger. Food Chem 2015; 173: 156-162
  PubMedSearch in Google Scholar
• 13 Hao W, Chen Z, Yuan Q, Ma M, Gao C, Zhou Y, Zhou H, Wu X, Wu D, Farag MA, Wang S, Wang Y. Ginger polysaccharides relieve ulcerative colitis via maintaining intestinal barrier integrity and gut microbiota modulation. Int J Biol Macromol 2022; 219: 730-739
  PubMedSearch in Google Scholar
• 14 Wang Y, Wang S, Song R, Cai J, Xu J, Tang X, Li N. Ginger polysaccharides induced cell cycle arrest and apoptosis in human hepatocellular carcinoma HepG2 cells. Int J Biol Macromol 2019; 123: 81-90
  PubMedSearch in Google Scholar
• 15 Aggarwal BB. Nuclear factor-κB: The enemy within. Cancer Cell 2004; 6: 203-208
  PubMedSearch in Google Scholar
• 16 Mantovani A. Inflammation by remote control. Nature 2005; 435: 752-753
  PubMedSearch in Google Scholar
• 17 Cao J, Tang D, Wang Y, Li X, Hong L, Sun C. Characteristics and immune-enhancing activity of pectic polysaccharides from sweet cherry (Prunus avium). Food Chem 2018; 254: 47-54
  PubMedSearch in Google Scholar
• 18 Wang P, Liao W, Fang J, Liu Q, Yao J, Hu M, Ding K. A glucan isolated from flowers of Lonicera japonica Thunb. inhibits aggregation and neurotoxicity of Aβ42. Carbohydr Polym 2014; 110: 142-147
  PubMedSearch in Google Scholar
• 19 Zhang S, Zhang C, Li M, Chen X, Ding K. Structural elucidation of a glucan from Crataegus pinnatifida and its bioactivity on intestinal bacteria strains. Int J Biol Macromol 2019; 128: 435-443
  PubMedSearch in Google Scholar
• 20 Lam JH, Baumgarth N. Toll-like receptor mediated inflammation directs B cells towards protective antiviral extrafollicular responses. Nat Commun 2023; 14: 3979
  PubMedSearch in Google Scholar
• 21 Benalaya I, Alves G, Lopes J, Silva LR. A review of natural polysaccharides: Sources, characteristics, properties, food, and pharmaceutical applications. Int J Mol Sci 2024; 25: 1322
  PubMedSearch in Google Scholar
• 22 Ji X, Cheng Y, Tian J, Zhang S, Jing Y, Shi M. Structural characterization of polysaccharide from jujube (Ziziphus jujuba Mill.) fruit. Chem Biol Technol Agric 2021; 8: 54
  PubMedSearch in Google Scholar
• 23 Ji X, Guo J, Ding D, Gao J, Hao L, Guo X, Liu Y. Structural characterization and antioxidant activity of a novel high-molecular-weight polysaccharide from Ziziphus Jujuba cv. Muzao. J Food Meas Charact 2022; 16: 2191-2200
  PubMedSearch in Google Scholar
• 24 ShanChen. Khan BM, Cheong KL, Liu Y. Pumpkin polysaccharides: Purification, characterization and hypoglycemic potential. Int J Biol Macromol 2019; 139: 842-849
  PubMedSearch in Google Scholar
• 25 Yang X, Li A, Li X, Sun L, Guo Y. An overview of classifications, properties of food polysaccharides and their links to applications in improving food textures. Trends Food Sci Tech 2020; 102: 1-15
  PubMedSearch in Google Scholar
• 26 Li Y, Zhang X, Li Y, Yang P, Zhang Z, Wu H, Zhu L, Liu Y. Preparation methods, structural characteristics, and biological activity of polysaccharides from Salvia miltiorrhiza: A review. J Ethnopharmacol 2023; 305: 116090
  PubMedSearch in Google Scholar
• 27 Wang Y, Zhang H. Advances in the extraction, purification, structural-property relationships and bioactive molecular mechanism of Flammulina velutipes polysaccharides: A review. Int J Biol Macromol 2021; 167: 528-538
  PubMedSearch in Google Scholar
• 28 Lin X, Xiao B, Liu J, Cao M, Yang Z, Zhao L, Chen G. An acidic heteropolysaccharide rich in galactose and arabinose derived from ginger: Structure and dynamics. Food Biosci 2023; 56: 103127
  PubMedSearch in Google Scholar
• 29 Li Z, Du Z, Wang Y, Feng Y, Zhang R, Yan X. Chemical Modification, Characterization, and Activity Changes of Land Plant Polysaccharides: A Review. Polymers (Basel) 2022; 14: 4161
  PubMedSearch in Google Scholar
• 30 Sun L, Wang L, Zhou Y. Immunomodulation and antitumor activities of different-molecular-weight polysaccharides from Porphyridium cruentum . Carbohydr Polym 2012; 87: 1206-1210
  PubMedSearch in Google Scholar
• 31 Yin M, Zhang Y, Li H. Advances in research on immunoregulation of macrophages by plant polysaccharides. Front Immunol 2019; 10: 145
  PubMedSearch in Google Scholar
• 32 Coutinho-Wolino KS, Almeida PP, Mafra D, Stockler-Pinto MB. Bioactive compounds modulating Toll-like 4 receptor (TLR4)-mediated inflammation: pathways involved and future perspectives. Nutr Res 2022; 107: 96-116
  PubMedSearch in Google Scholar
• 33 Ji M, Sun L, Zhang M, Liu Y, Zhang Z, Wang P. RN0D, a galactoglucan from Panax notoginseng flower induces cancer cell death via PINK1/Parkin mitophagy. Carbohydr Polym 2024; 332: 121889
  PubMedSearch in Google Scholar
• 34 Wang P, Zhang L, Yao J, Shi Y, Li P, Ding K. An arabinogalactan from flowers of Panax notoginseng inhibits angiogenesis by BMP2/Smad/Id1 signaling. Carbohydr Polym 2015; 121: 328-335
  PubMedSearch in Google Scholar
• 35 Cong Q, Chen H, Liao W, Xiao F, Wang P, Qin Y, Dong Q, Ding K. Structural characterization and effect on anti-angiogenic activity of a fucoidan from Sargassum fusiforme . Carbohydr Polym 2016; 136: 899-907
  PubMedSearch in Google Scholar

• Supplementary Material