Astragalus polysaccharides: structure-immunomodulation relationships, multi-target pharmacological activities, and cutting-edge applications in immune modulation

Abstract

Astragalus Polysaccharide (APS), the primary bioactive component of Astragalus, exhibits multi-faceted immunomodulatory properties. Its efficacy stems not from broad, non-specific stimulation but from the precise engagement of a network of cell surface immune receptors. This review synthesizes the critical structure-immunomodulatory network relationship of APS, positioning Toll-like receptor 4 as a central mediator. Key insights reveal that APS bioactivity is governed by a specific molecular weight window, critical monosaccharide ratios, and distinct glycosidic linkages. These structural features enable APS to interact with TLR4, potentially in collaboration with other pattern recognition receptors such as the Mannose Receptor and Dectin-1, to initiate integrated signaling. Future research must prioritize multi-omics and structural biology to map precise receptor-binding sites, establish robust standardization and quality control protocols, and advance translational clinical studies for APS-based adjuvant development. This work provides a strategic framework for advancing APS from a traditional remedy into a novel, mechanism-driven immunomodulatory agent.

1. Introduction

Plant-derived polysaccharides constitute a major class of biological response modifiers, renowned for their ability to interact with the immune system to either enhance host defenses or restore immunological homeostasis (1, 2). Among these, Astragalus polysaccharide (APS), a key bioactive component from Astragalus membranaceus, has garnered significant attention due to its demonstrated immunomodulatory, antitumor, anti-inflammatory, and metabolic-regulatory properties (3). A particularly important feature distinguishing APS from broad-spectrum immune stimulants is its ability to mediate effects through precise interactions with immune components, facilitating a finely-tuned regulatory response (4). However, this precision is complicated by a fundamental challenge: the substantial structural heterogeneity of APS, which includes variations in molecular weight, monosaccharide composition, glycosidic linkage patterns, and tertiary conformation. Therefore, a critical challenge in the field lies in deciphering how these specific structural features govern immune recognition and initiate downstream signaling, a knowledge gap that must be addressed for its rational development as a targeted immunotherapeutic agent.

The immunomodulatory capacity of APS is primarily attributed to its engagement with pattern recognition receptors (PRRs) (5). While Toll-like receptor 4 (TLR4) is the most extensively characterized receptor, serving a central role in activating the MyD88-dependent pathway, it is crucial to recognize that immune regulation inherently involves coordinated signaling across an integrated network (6–8). In line with this concept, mounting evidence suggests that other PRRs, including the mannose receptor (MR) and Dectin-1, may also function as targets for APS, an inference based on its monosaccharide profile and structural similarities to established ligands (9–11). Despite these indications, direct experimental validation of these interactions and a comprehensive delineation of the multi-receptor signaling landscape remain elusive, representing a considerable knowledge gap.

Although prior research has demonstrated the immunomodulatory effects of APS, highlighting their broad impact on immune organs, cells, and related diseases such as cancer and infections (12), a significant limitation of this existing body of work is that it has primarily emphasized phenomenological descriptions of immune responses. Specifically, it has not sufficiently delved into the structural basis of APS bioactivity, leaving a critical gap in understanding how specific molecular features (e.g., molecular weight, monosaccharide composition, glycosidic linkages) govern immune receptor engagement and signaling pathways. Furthermore, earlier reviews often lack integration of multi-target pharmacological mechanisms and cutting-edge applications, such as nanocarrier systems or vaccine adjuvants, which are crucial for translational progress. In contrast, this review is designed to address these limitations by systematically elucidating the structure-immunomodulation relationship of APS, positioning TLR4 as a central mediator and validating this interaction via modern analytical techniques. We further expand the scope by detailing multi-target activities beyond immunomodulation, including metabolic and anti-tumor effects, alongside innovative delivery strategies, thereby providing a strategic framework for advancing APS as a precision-based immunotherapeutic agent. This integrated approach not only clarifies the mechanistic foundation but also underscores the potential of APS in novel clinical applications, representing a significant advance in the field.

2. The influence of processing on the structure of Astragalus polysaccharides

Heterogeneity is the most distinctive feature of APS, arising principally from variations in the plant’s geographic origin, growth duration, post-harvest processing, and the specific extraction and purification protocols employed. The geographical origin of the Astragalus plant is a critical determinant of the resulting polysaccharide’s structural properties. Research comparing Astragalus from various producing areas (such as Inner Mongolia, Shanxi, and Gansu) reveals that although the fundamental monosaccharide profile and predominant glycosidic bond types of the water-soluble APS are similar, they exhibit marked variations in molecular weight (Mw), polydispersity, and the methylation level of uronic acid residues (13). These parameters are regarded as critical markers for differentiating APS based on their geographic provenance. Concurrently, these structural differences are thought to be linked to regional environmental conditions, including solar radiation, rainfall, and soil constituents. These factors modulate the enzymatic machinery responsible for polysaccharide biosynthesis in Astragalus, thereby inducing structural modifications in the final polysaccharide product (14). For example, Sheng et al. (13) conducted a systematic comparison of the structural profiles of Astragalus polysaccharides from various geographical sources, revealing a strong correlation between these structural disparities and biological activities, including antioxidant effects. Similarly, Zhang and colleagues (15) separated two homogeneous neutral polysaccharide fractions, designated APS-I (120 kDa) and APS-II (12 kDa). Both were identified as heteropolysaccharides with a high glucose content (exceeding 90%), but structural analysis revealed that APS-II adopted a highly branched architecture with a compact, spherical conformation, in contrast to the less branched, more extended linear chain morphology of APS-I. Capitalizing on these distinct features, the investigators evaluated their antitumor immunomodulatory activities, finding APS-II more potent at the same dosage.

Beyond geographic and cultivar variations, traditional Chinese medicine processing techniques significantly impact APS structure and activity. For instance, honey-frying causes profound changes, with studies confirming that honey-fried Astragalus polysaccharide (HAPS3a) undergoes significant structural alterations compared to raw APS (APS3a) (16). These alterations primarily include: (1) Reduced molecular weight due to partial hydrolysis of glycosidic bonds during processing (17); (2) Altered monosaccharide composition ratios from degradation or transformation (17, 18); and (3) Chemical modifications like Maillard reactions, introducing new functional groups and enhancing intermolecular hydrogen bonding, which affect solubility and conformation (17). These changes are considered the material basis for enhanced efficacy in functions like “tonifying Qi” and “moistening the lungs.” For example, honey-processed APS (HAPS) shows superior anti-inflammatory efficacy in murine colitis models (16). This phenomenon is not unique to APS; similar changes in monosaccharide composition and immune activity have been observed in other herbs like Polygonum multiflorum after processing (19). Collectively, these findings indicate that processing modulates bioactivity by precisely altering the primary structure of polysaccharides, providing a molecular perspective for understanding the mechanism of traditional processing. Other methods, such as drying, can also induce polymer aggregation or partial degradation, further underscoring that every step from field to final product shapes the structural and therapeutic properties of APS (20, 21).

Furthermore, extraction and purification methods contribute to APS heterogeneity. Techniques such as hot water extraction, ultrasound-assisted, microwave-assisted, or enzyme-assisted extraction, along with purification steps like alcohol precipitation and chromatography, selectively enrich polysaccharides with varying molecular weights, charges, or solubilities, leading to distinct structural characteristics (3, 22, 23). This highlights the critical need to standardize and document the entire workflow for reliable structure-activity relationship (SAR) studies. The relationship between different processing/extraction methods of APS and the structure and activity are summarized in Figure 1.

Figure 1.

Processing methods and structure-activity relationship of Astragalus polysaccharides.

3. Structure-immunomodulation relationships

The innate immune system, which functions as the host’s primary defense against pathogens, is centered on pattern recognition receptors (PRRs) that detect pathogen-associated molecular patterns (PAMPs) (24–26). Among these PRRs, receptors such as Toll-like receptors (TLRs) and C-type lectin receptors (CLRs) act as immunological “sentinels” that perceive external danger signals (27). Substantial evidence indicates that APS, as a typical plant polysaccharide, is recognized by these sentinels; it initiates its immunomodulatory activity by binding to PRRs, thereby mimicking PAMPs to trigger downstream immune signaling cascades (28, 29). The molecular mechanism of APS immune regulation is shown in Figure 2.

Figure 2.

Molecular mechanism of immunomodulation by polysaccharides from Astragalus.

3.1. Interaction of Astragalus polysaccharides with toll-like receptor 4

Among various PRRs, TLR4 is the most extensively studied and well-evidenced key receptor mediating the immunomodulatory effects of Astragalus polysaccharides. This receptor complex, comprising TLR4 and its accessory protein MD-2, is the principal sensor for Gram-negative bacterial lipopolysaccharide (LPS) and is pivotal in innate immune activation (30, 31). Accordingly, research confirms that APS functions as a TLR4 ligand, initiating downstream signaling cascades. Shao et al. (32) studies demonstrating that APS significantly upregulates TLR4 expression on immune cells such as macrophages and dendritic cells. A key piece of evidence is that anti-TLR4 monoclonal antibodies inhibit APS binding to macrophages, offering compelling support for a direct interaction.

Numerous studies consistently demonstrate that APS significantly upregulates TLR4 expression on the surface of immune cells such as macrophages and dendritic cells, activates the TLR4-mediated MyD88-dependent signaling pathway, leads to the recruitment of adaptor proteins MyD88 and TRAF-6, and activates transcription factors NF-κB and activator protein-1 (AP-1) (8, 33, 34). Following its binding to TLR4, APS initiates downstream signal transduction by recruiting the adaptor protein MyD88. MyD88 then sequentially activates interleukin-1 receptor-associated kinases (IRAKs) and tumor necrosis factor receptor-associated factor 6 (TRAF6), leading to the assembly of a signaling complex (26, 35). This signaling complex subsequently activates downstream mitogen-activated protein kinases (MAPKs), such as p38, ERK, and JNK, as well as the IκB kinase (IKK) complex. The activation of IKK catalyzes the phosphorylation and proteasomal degradation of the inhibitory protein IκBα, which liberates and activates the pivotal transcription factors NF-κB and AP-1 (36, 37). These activated transcription factors then translocate to the nucleus, driving the expression of pro-inflammatory cytokines and immunomodulatory molecules such as TNF-α, IL-6, IL-1β, and nitric oxide (NO) (38, 39).

It is important to emphasize that the majority of TLR4 involvement in APS activity is inferred from downstream signaling changes, antibody-blocking experiments, and receptor-expression modulation; direct biophysical confirmation of APS–TLR4/MD-2 binding (e.g., SPR, ITC) remains limited to date. Therefore, while the status of TLR4 as a core target of APS is well-established, it is crucial to note that current research largely relies on indirect measurements of downstream signaling to infer its role. Consequently, the critical questions of which specific structural features in APS mimic LPS to bind the TLR4/MD2 complex, and the associated binding affinity/kinetics, remain key subjects for deeper investigation.

3.2. Interaction between Astragalus polysaccharides and mannose receptor

The extracellular region of MR contains multiple tandem C-type lectin-like domains (CTLDs), with CTLD-4 and CTLD-5 being the key regions for binding carbohydrate ligands (40, 41). These domains act in concert to facilitate high-affinity binding to polysaccharide structures that possess multiple terminal mannose residues (42). It is well-established that numerous polysaccharides originating from fungi, yeast, and certain plant species exert their immunomodulatory effects via engagement with the MR. For instance, mannan from Candida albicans and specific polysaccharides from Ganoderma lucidum, both rich in mannose, have been identified as recognition ligands for the MR (6, 43, 44); this engagement can trigger cellular endocytosis, antigen presentation, and cytokine modulation (45). Given the presence of mannose in APS, it is a plausible candidate for MR interaction. Chemical analyses of APS reveal a heterogeneous monosaccharide composition that includes a significant proportion of mannose (9), providing a structural basis for this potential interaction. Supporting this, research has demonstrated that APS treatment upregulates the cell surface expression of the MR (10). This may be related to the direct or indirect activation of cells by APS, whereby MR expression is upregulated as a downstream effect of cell activation to enhance the cell’s ability to recognize other pathogens (46). However, the observed upregulation in expression is insufficient to establish MR as a direct receptor for APS; this could also be a downstream effect of general cell activation. This considerable knowledge gap delineates a clear research direction. Employing modern techniques to systematically validate the APS-MR interaction is therefore imperative, as it will not only refine our understanding of the immunomodulatory mechanism of APS but could also pioneer new pathways for developing highly targeted polysaccharide immunomodulators.

3.3. Interaction of Astragalus polysaccharides with dectin-1

Dectin-1, a member of the C-type lectin receptors (CLRs) family, is the principal receptor for fungal β-glucans, characterized by a β-(1,3)-linked backbone often with β-(1,6)-linked side chains (47–49). Recognition occurs via the extracellular C-type lectin-like domain (CTLD) in a calcium-independent manner, where the glucan chain interacts with a hydrophobic pocket (50–52). Ligand binding induces receptor clustering, phosphorylation of the intracellular ITAM-like motif, and recruitment of spleen tyrosine kinase (Syk) (53, 54). Activated Syk propagates the signal, leading to NF-κB activation via the CARD9-Bcl10-MALT1 complex, and can also elicit reactive oxygen species (ROS) production, NLRP3 inflammasome activation, and enhanced phagocytosis (55–57). A notable feature is the significant synergistic crosstalk that occurs between the Dectin-1 and Toll-like receptor (TLR, especially TLR2) signaling pathways. Simultaneous activation of both receptors leads to a more robust production of cytokines like TNF-α and IL-1β, facilitating a finely tuned immune response (29, 58).

Given the structural specificity of APS, Dectin-1 represents a promising potential target, particularly due to the presence of glucose configured into β-glucan-like structures. It is therefore postulated that APS may achieve synergistic amplification of its effects by simultaneously engaging both TLR4 and Dectin-1 receptors. However, while the involvement of TLR4 is well-documented, current research faces significant mechanistic gaps. Direct experimental evidence, such as quantitative binding affinity data for the APS-TLR4/MD-2 interaction or confirmation of binding to Dectin-1, is currently absent. This lack of direct evidence means the precise molecular patterns recognized by these receptors and the potential for synergistic signaling remain unclear. To address these gaps and construct a complete APS-PRR interaction map, future research should focus on a multi-faceted approach. First, obtaining structurally defined, homogeneous APS fractions through advanced separation techniques is essential. Subsequently, direct binding affinities (e.g., Kd values) and kinetics for receptors like TLR4/MD-2 and Dectin-1 must be quantified using biophysical methods like Surface Plasmon Resonance (SPR). These efforts should be combined with molecular docking simulations and site-directed mutagenesis to elucidate binding epitopes at an atomic level. Finally, systematic comparisons of well-characterized APS fractions in cellular models, using specific inhibitors or gene-knockout cells, are needed to delineate the individual and synergistic contributions of different PRR pathways to APS’s overall immunomodulatory effect. This comprehensive strategy is critical for advancing APS from a complex mixture to a precisely defined immunomodulatory agent.

3.4. Immune microenvironment reprogramming

Immune reprogramming” describes the process of altering immune cell identity and function through external intervention (59), providing a framework for natural product-based therapy. Research indicates that APS acts not as a simple immune booster but as a precise modulator that reprograms the immune microenvironment. APS directly or indirectly activates macrophages, dendritic cells, and other immune cells, binding to their surface receptors. This interaction promotes the proliferation and differentiation of immune cells, increases cytokine secretion, and alleviates immune suppression (Figure 3). These results provide a relevant evidence base for guiding the use of polysaccharides as adjuvants in cancer immunotherapy.

Figure 3.

The relationship between macrophages, dendritic cells, T cells, and cytokines in the presence of APS.

3.4.1. Reversal of T cell exhaustion

In recent years, cancer immunotherapy, represented by Immune Checkpoint Inhibitors (ICIs) and Chimeric Antigen Receptor T-cell (CAR-T) therapy, has achieved revolutionary breakthroughs. However, a significant clinical challenge remains: a substantial subset of patients either fails to respond initially or acquire resistance. A pivotal mechanism for this limitation is T cell exhaustion (60, 61), which is intimately linked to the profoundly immunosuppressive nature of the tumor microenvironment (TME). APS can remodel the TME to counteract this exhaustion through multiple mechanisms. First, it modulates key immunosuppressive cell populations. Research demonstrates that APS can alter the differentiation and function of myeloid-derived suppressor cells (MDSCs) (62) and potentially attenuate regulatory T cell (Treg) recruitment, possibly via inhibition of Foxp3 expression (63). Diminishing the abundance and suppressive activity of these cells alleviates the burden on exhausted T cells, fostering a conducive milieu for functional recovery. Second, APS enhances antigen presentation. It potently promotes the maturation and functional capacity of dendritic cells (DCs), consequently boosting their antigen presentation ability (33, 64). Fully mature DCs deliver more robust T cell activation signals, a process that may promote the differentiation of T cells into effector phenotypes rather than exhausted states. Supporting this, studies in animal models indicate that co-administration of APS with chemotherapy drugs markedly enhances the infiltration of CD8⁺ T cells into tumor sites (65), shifting the balance between effector and suppressor cells. Enhanced recruitment of effector cells to the tumor site is a fundamental prerequisite for antitumor efficacy and likely shifts the balance between effector and suppressor cells within the TME, potentially overcoming the local immunosuppression.

A particularly compelling mechanism involves the direct targeting of the PD-1/PD-L1 pathway, a cornerstone of T cell exhaustion. An illuminating study revealed that APS can stimulate the endogenous generation of antibodies targeting PD-1. These antibodies inhibit the PD-1/PD-L1 interaction, leading to suppressed tumor progression (66). This suggests a novel paradigm where APS acts as a molecular mimic, eliciting a humoral response to indirectly facilitate checkpoint inhibition. Furthermore, other studies report that APS can directly reduce the expression levels of PD-L1 within the TME (33). Downregulation of this key ligand effectively diminishes the inhibitory signals received by T cells, thereby contributing to the relief from chronic exhaustion.

3.4.2. Modulation of regulatory T cells

Regulatory T cells (Tregs), defined by the master transcription factor Foxp3, constitute a pivotal immunosuppressive subset essential for maintaining self-tolerance and controlling inflammation (67). However, their hyperactivation in settings like cancer suppresses productive immune responses, facilitating immune evasion (68, 69). The modulatory impact of APS on Tregs demonstrates a remarkable “context-dependency,” performing opposing immunoregulatory roles to restore immune homeostasis. In the context of cancer and immunosuppression, APS primarily acts to inhibit Treg function. For instance, APS has been reported to suppress the functionality of CD4⁺CD25high Treg cells in the microenvironment of human liver cancer (68). Similarly, research indicates that APS promotes tumor regression by inhibiting Treg activation (69, 70). This dampening of Treg-mediated suppression effectively “unleashes” cytotoxic T lymphocytes (CTLs), enhancing anti-tumor efficacy. Supporting this mechanism in a non-cancer context, APS was found to improve the host’s immunosuppressed state in a burn-induced sepsis model by inhibiting the negative immunomodulatory function of CD4⁺CD25high T cells, thereby mitigating septic progression (71). In these scenarios, the net result is the reversal of immunosuppression.

Conversely, under conditions of excessive immune activation and tissue injury, APS can promote Treg activity to curb inflammation. A study demonstrated that APS markedly ameliorated experimental colitis in rats by promoting the generation of Treg cells within the intestinal Peyer’s patches (72). Furthermore, APS may also reprogram Treg functionality; in a periodontitis model, it reduced the abundance of Foxp3⁺ Tregs while increasing the population of IL-10-producing Tregs, suggesting a sophisticated form of regulation that enhances anti-inflammatory capacity (73). Here, the fundamental outcome is the re-establishment of immune tolerance, positioning APS as an “immune stabilizer”.

3.5. Other potential receptors

NOD-like receptors (NLRs) are a class of cytosolic pattern recognition receptors (PRRs) characterized by a typical structure: an N-terminal effector domain, a central nucleotide-binding domain (NACHT), and a C-terminal leucine-rich repeat (LRR) domain responsible for ligand sensing (74). Among them, NLRP3 is a well-studied member activated by a wide array of stimuli, from pathogens to endogenous danger signals (75). A key mechanism underlying the immunomodulatory effects of APS appears to be the direct or indirect modulation of NLR signaling pathways, particularly the NLRP3 inflammasome, providing a molecular basis for its anti-inflammatory properties. This is evidenced by studies across different inflammatory disease models. In a mouse model of DSS-induced inflammatory bowel disease, APS significantly inhibited NLRP3 inflammasome activation, leading to reduced levels of mature IL-1β and IL-18 (76). Similarly, in an ovalbumin-induced allergic rhinitis model, APS alleviated nasal inflammation by suppressing NLRP3 inflammasome activation (77). The inhibitory action of APS is thought to operate at multiple levels of the NLRP3 activation cascade. Proposed mechanisms include the scavenging of reactive oxygen species (ROS)—a key trigger for NLRP3—through its antioxidant properties, the modulation of mitochondrial function, or direct interference with protein interactions within the inflammasome complex. However, the precise molecular targets and detailed mechanisms remain an important area for future investigation.

3.6. Structural features dictating immunological activity

The biological activity of APS is profoundly influenced by their specific chemical structural attributes, including molecular weight, monosaccharide composition, types of glycosidic linkages, branching architecture, and three-dimensional conformation (Figure 4). However, owing to the inherent structural complexity of APS, investigations explicitly correlating its structure with immunological activity remain limited. Nevertheless, synthesizing insights from the existing body of research is crucial for guiding future studies. Therefore, this section provides a discussion of the relationship between APS structure and its immunomodulatory effects, drawing upon available findings. The relationship between the structure of APS and immune regulatory activity is summarized in Table 1.

Figure 4.

The biological activities of Astragalus polysaccharide.

Table 1.

Molecular weight, monosaccharide compositions and structural characteristics of Astragalus polysaccharides.

Fraction	Mw (kDa)	Monosaccharide composition (molar ratio)	Structural features	Mechanism	Biological activities	Ref
APS-I	>500	Glc: Gal: Ara: Rha: GalA=1.5:1:5.4:0.08:0.1	D-Glcp-(1→, →4)-D-Glcp-(1→, →2)-L-Rhap-(1→, D-Araf-(1→, →5)-D-Araf-(1→, →2,5)-D-Araf-(1→, →4)-D-Galp-(1 →	The phagocytic ability of macrophages, splenic lymphocyte proliferation, spleen T-lymphocyte proliferation, enhancing the toxic activity of NK cells (mouse, in vitro), spleen T-lymphocyte subsets CD3+ level significantly increased, and the CD4+/CD8+ level decreased, increased spleen and thymus indices	Immunomodulatory activity	(78)
APS-II	10	Glc:Gal:Ara:Rha:GalA=9:1:1.4:0.04:0.001	α-D-Glcp-(1→, →4)-α-D-Glcp-(1→, →6)-α-D-Glcp-(1→, →4,6)-α-D-Glcp-(1→, →3,4,6)-α-D-Glcp-(1→, → 2)-α-L-Rhap-(1→, α-D-Araf-(1→, →5)-α-D-Araf-(1→, →4)-β-D-Galp-(1 →	The phagocytic ability of macrophages, splenic lymphocyte proliferation, spleen T-lymphocyte proliferation, enhancing the toxic activity of NK cells (mouse, in vitro), spleen T-lymphocyte subsets CD3+ level significantly increased, and the CD4+/CD8+ level decreased, increased spleen and thymus indices	Immunomodulatory activity	(78)
AX-I-3b	7.87	Ara: Xyl: Glu=10.4:79.3:1.1	→2,3,4)-β-D-Xylp-(1→, →4)-β-D-Arap-(1→, →4)-β-D-Glcp-(1→	the organ index of spleen and thymus increased, Elevated levels of cytokines IL-2, IL-6, and TNF - α in mouse serum, The proportion of CD4+T cells and CD8+T cells in peripheral blood of mice increases, Increased expression of p53 in tumor cells upregulation of Bax/Bcl-2 ratio in tumor cells,	Antitumor and Immunological Activities	(79)
AMA-1-b-PS2	–	Ara: Fuc: Gal: Glu: Man: Rha: Xyl: GalA: GluA: GlcA=12.8:4.5:25.6:23.6:24.8:5.1: 0.7:1.5:1.5:1.4	β-D-(1→3)-galactans	Increased Peyer’s patch cells	Intestinal immune system-modulating	(80)
APSII	11.4	Xyl: Glu: Ara: Rha: Man: Gal=9.22:77.89:1:5.18:4.54:2.17	–	Increase the expression of surface markers MHCII, CD40, CD80, and CD86 on RAW264.7, induce NO production, and enhance cell phagocytic ability	Immunoregulatory effects	(81)
APS-IV	20.7	Glu	(1→4)-linked backbone with a (1→6)-linked branch every 10 residues	Stimulating the activity of mouse B cells without promoting T cell proliferation, Promote proliferation of spleen cells in BABL/c mice	Immunomodulatory activity	(82)
AMP	920.7	Glu: Ara: Gal=91.0:6.2:2.8	–	Stimulate RAW264.7 cells to produce NO and upregulate iNOS, TNF - α, IL-1 β, IL-6 mRNA expression through NF - κ B and MAPKs pathways	Immunomodulatory activity	(83)
RAP	1334	Rha: Ara: Glu: Gal: GalA=0.03:1.00:0.27:0.36:0.30	Backbone consists of →1,2,4)- Rhap, →1,4)-α- Glcp, →1,4)-α- GalAp6Me, →1,3,6)-β- Galp, with branched at O-4 of the →1,2,4)- Rhap and O-3 or O-4 of →1,3,6)-β- Galp	In vitro promotion of IL-1 β, TNF - α, IL-10, IL-12, and GM-CS production in human PBMCs	Immunomodulating effects	(84)
cAMPs-1 A	12.3	Fuc: Ara: Gal: Glu: Xyl=0.01:0.06:0.20:1.00:0.06	–	improve the macrophages pinocytosis and NK cells killing activity, Elevated proportion of CD4+and CD8+T cells in peripheral blood of tumor bearing mice,	Antitumor and immunomodulatory effects	(85)
ASP	2.1	Ara: Gal: Glu: Man=1.00:0.98:3.01:1.52	pyranose ring and α-type glycosidic linkages	Enhance the serum cytokine levels of H22 liver cancer cells, significantly inhibit their growth (TNF - α, IL-2, and IFN - γ) and immune cell activity (macrophages, lymphocytes, and NK cells)	Antitumor activity	(86)
APS-I	>2,000	Rha: GalA: Gal: Glu: Ara= 0.1:0.39:13.4:17.2:1	→3)-D-Glc-(1→, →4)-D-Glc-(1→, →6)-D-Gal-(1→, →3,4)-D-Gal (1→	Increase spleen and thymus indices	Immunomodulatory activity	(87)
APS-II	10.2	Rha: GalA: Gal: Glu: Ara= 0.14:0.14:9.6:24.04:1	D-Glcp-(1→, →4)-D-Glc-(1	Increase spleen and thymus indices, phenylalanine metabolism, cysteine and methionine metabolism, tricarboxylic acid cycle (TCA cycle) and arginine and proline metabolism	Immunomodulatory activity	(87)
APS-III	0.286	Rha: GalA: Gal: Glu: Ara= 0.375:0.375:18.8:90.5:1	→4)-D-Glc-(1→	Increase spleen and thymus indices	Immunomodulatory activity	(87)
APS-I	120	Glu: Gal: Man: Ara	–	TLR4-MyD88-NF-κB signaling pathway	Anti-tumor immunomodulatory effects	(15)
APS-II	12	Glu: Gal: Man: Ara	–	TLR4-MyD88-NF-κB signaling pathway	Anti-tumor immunomodulatory effects	(15)
APS1	106.5	Man: Rha: GalA: Glu: Gal: Ara= 0.79:1.70:3.28:7 9.07:6.63:8.56	→1,4)-β-Galp occasionally substituted by α-Araf at O-2 and/or O-3	Increase phagocytic activity and cytokine IL-6 and IL-1 β mRNA expression in RAW264.7 cells, MAPKs and NF-κB signaling pathways, promote the expression of IL-6, IL-1β and TNF-α	Immunomodulatory activities	(88)
APS2	114.5	Man: Rha: GalA: Glu: Gal: Ara= 1.61:6.05:10.21:6.34:27.39:48.39	long branches of α-Araf, which were attached to the O-3 of →1,6)-β-Galp of the backbone	Increase phagocytic activity and cytokine IL-6 and IL-1 β mRNA expression in RAW264.7 cells, MAPKs and NF-κB signaling pathways, promote the expression of IL-6, IL-1β and TNF-α	Immunomodulatory activities	(88)
APSN	4.97	Glu	a highly branched glucan with a →1,4)-α-D-glucopyranosyl main chain and side chains at the O-6 position	Inhibition of NF-κB pathway in RAW 264.7 macrophages promote endothelial cell proliferation and angiogenesis, and expression of VEGFA, EGF, and EGFL6	Immunomodulatory activities and anti-inflammatory activity	(89)

3.6.1. Molecular weight

Molecular weight (Mw) is a fundamental property dictating polysaccharide bioactivity, primarily by modulating physicochemical characteristics such as solubility and viscosity, which in turn influence absorption and bioavailability. Evidence suggests that the most potent immunostimulatory polysaccharides often fall within a specific Mw window, potentially due to optimal solubility, tissue diffusibility, and capacity for productive receptor clustering. The relationship between Mw and activity has been systematically demonstrated for APS. Li and colleagues (90) fractionated crude APS into three distinct Mw ranges: a high-Mw fraction (>2000 kDa, APS-I), a medium-Mw fraction (~10 kDa, APS-II), and a low-Mw fraction (~300 Da, APS-III). Comparative analysis revealed a non-linear, parabolic relationship, identifying APS-II as the most immunomodulatory fraction. In vivo studies in an immunosuppressed mouse model showed APS-II was most effective at restoring immune organ indices and potentiating immune responses. It significantly outperformed other fractions in promoting splenic lymphocyte proliferation, inducing Th1-type cytokines (IL-2, IFN-γ), and enhancing innate immunity via macrophage phagocytosis and NK cell cytotoxicity (91, 92). The superior potency of the ~10 kDa fraction is attributed to its optimal physicochemical properties, such as good aqueous solubility and low viscosity, which promote systemic absorption and efficient engagement with immune cells (93, 94). In contrast, APS-I (>2000 kDa) showed weaker activity due to poor solubility and high viscosity, while APS-III (<1 kDa) exhibited minimal activity, suggesting a minimal chain length and specific 3D architecture are prerequisites for immune activation (87) (Table 2).

Table 2.

Significant differences in the structure and activity of different Mw components of Astragalus polysaccharides.

Name	Mw	Monosaccharide composition	Immune activity intensity	Key evidence	Ref
APS-I	>2000 kDa	High Glc (Gal: Glc=17.2:1)	Medium	Promote macrophage phagocytosis, but weak cytokine induction	(87)
APS-II	10 kDa	High Gal (Gal: Glc =14.9:1), low GalA	optimal	NK cell activation, T/B cell proliferation, and IgG secretion
APS-III	<1 kDa	extremely high Glc (Glc: Ar=90.5:1 a)	weak	Mainly focused on metabolic regulation, with limited immune activity

This systematic comparison across orders-of-magnitude different fractions in APS research provides a clear functional differentiation that is less commonly encountered in studies of other polysaccharides like Lentinan. While Lentinan’s bioactivity is also known to relate to its molecular weight, extraction method, and conformation (95). The reported molecular weight range for Lentinan is broad, spanning from tens of kDa to several million Da; for example, studies have isolated fractions of 25.5 kDa, 306.2 kDa, and 605.4 kDa (96), or components as high as 6.8 x 10⁶ g/mol (97). The research focus for Lentinan differs, emphasizing the elucidation of its binding to specific receptors (e.g., Dectin-1) and subsequent signaling pathways (98, 99). Furthermore, the investigation of the low molecular weight APS fraction expands the understanding of small sugar bioactivity, an aspect often overlooked in traditional polysaccharide studies focused on high Mw polymers.

Therefore, while an optimal Mw range for APS activity is evident, its precise delineation remains tentative. The current suggestion of an optimal range (e.g., ~10 kDa) is inferred from limited comparisons, and a universal “optimal molecular weight window” for APS remains to be conclusively defined.

3.6.2. Monosaccharide composition

A significant correlation exists between the immunomodulatory activity of APS and its specific monosaccharide composition. Although APS is a heterogeneous mixture, its bioactivity is critically determined by the quantitative ratios of its constituent monosaccharides, such as glucose, galactose, and arabinose. This principle is empirically supported by several studies. For instance, Guo et al. (9) demonstrated a positive correlation between the content of mannose, glucose, xylose, and fucose and the level of nitric oxide (NO) release, offering direct evidence that monosaccharide profile is a pivotal determinant of APS immunopotency. Further reinforcing this, Jiang and colleagues (100) verified that APS2 (a homopolysaccharide of arabinose) and APS3 (composed of Rha, Glu, Gal, and Ara in a distinct molar ratio) exhibited differing immunomodulatory potencies despite both stimulating splenocyte proliferation. The distinct activities of APS2 and APS3, which possess different monosaccharide compositions, provide compelling evidence that this parameter is a key factor underlying the variation in APS bioactivity. This principle of composition-dependent activity underscores why the elucidation of structure-activity relationships (SAR) is a central challenge in polysaccharide research. The highly complex and variable monosaccharide composition of well-known immunomodulatory polysaccharides like Ganoderma lucidum polysaccharides (GLPs) presents a significant obstacle to defining precise SAR (101). In this context, the structural profile of APS—characterized by a more homogeneous composition dominated by glucose and arabinose—offers a comparatively advantageous model for deciphering the link between specific structural motifs and their corresponding biological activities.

3.6.3. Glycoside bond configuration

The bioactivity of a polysaccharide is critically determined by its structural features, most notably the types of glycosidic linkages and its degree of branching (102). For APS, a key structural motif has been identified: a glucan backbone consisting of α-(1→4)-linked D-glucose residues, which is ubiquitous in its bioactive fractions (103). Beyond the backbone, the presence and specific attachment points of branch chains are of paramount importance. Beyond the backbone, the presence and specific attachment points of branch chains are of paramount importance for immunological activity. A common structural pattern associated with high immunopotency is the presence of branch chains linked at the C6 position of the α-(1→4)-glucan backbone (104). The introduction of this branching enhances structural complexity, which is thought to modulate bioactivity by altering solubility, three-dimensional conformation, and the ability to engage in multipoint attachments with cellular receptors (105–107). Functional studies have correlated immunoactivity with the presence of specific glycosidic linkages, including, but are not limited to, →2,3)-α-L-Rha-(1→, →5)-α-L-Ara-(1→, →3,4)-β-D-Gal-(1→, →6)-β-D-Gal-(1→, →4)-α-D-Glu-(1→, and →3,4,6)-β-D-Glu-(1→ (90).

The structural logic of APS becomes clearer when contrasted with well-defined polysaccharides like Lentinan. Lentinan’s activity is attributed to a highly ordered structure—a β-(1→3)-glucan backbone with β-(1→6) branches—that forms a stable triple-helical conformation crucial for high-affinity binding to receptors like Dectin-1 (29, 108, 109). In contrast, the structure of APS exhibits significant “heterogeneity” and “complexity,” being a mixture of various linkage types encompassing both α- and β-configurations (86). This very complexity, however, constitutes its unique advantage. Unlike the specific, targeted action of β-glucans, the diverse structural motifs in APS can interact with a broader repertoire of immune receptors (e.g., TLR2, TLR4, MR), thereby enabling a “multi-pronged” mode of action that may result in a more comprehensive and balanced immunomodulatory outcome. Furthermore, this naturally complex mixture may better mimic physiological immune signals, regulating homeostasis in a more nuanced manner (110).

Notwithstanding this progress, a profound understanding of APS structure-activity relationships (SAR) remains challenging. The acquisition of homogeneous polysaccharide samples with defined structures, followed by comprehensive sequential and 3D structural analysis, continues to present a substantial obstacle that directly impedes a deeper mechanistic understanding.

4. Multi-target pharmacological activities beyond immunomodulation

Plant polysaccharides are natural macromolecules that have attracted widespread research interest due to their low toxicity and significant therapeutic potential. Among them, Astragalus polysaccharide is particularly notable for its diverse biological activities, which include hypoglycemic, anti-non-alcoholic fatty liver disease, anti-inflammatory, and anti-tumor properties (Figure 5). This multi-target therapeutic profile underscores the significant potential of APS for further pharmaceutical development and medical application.

Figure 5.

Relations between structure and immune effect of Astragalus polysaccharides.

4.1. Improvement of insulin resistance

Insulin resistance represents the central pathophysiological defect in T2DM. It has been demonstrated that APS effectively enhances insulin sensitivity and modulates glycemic homeostasis via several mechanisms independent of classical immune pathways. These mechanisms can be broadly categorized into direct insulin-sensitizing actions and broader metabolic improvements. A primary direct action is the potent activation of AMPK, a master regulator of cellular energy status. AMPK activation enhances glucose uptake and suppresses hepatic gluconeogenesis, consequently alleviating glucotoxicity (111, 112). Concurrently, APS augments insulin signaling by activating the Akt/GLUT4 axis to facilitate glucose disposal in peripheral tissues and by suppressing protein tyrosine phosphatase 1B (PTP1B), a key negative regulator of insulin receptor signaling (39, 113–115). Beyond direct signaling, APS elicits comprehensive metabolic improvements, particularly in the liver. It modulates the hepatic SIRT1-PGC-1α/PPARα-FGF21 signaling cascade to enhance fatty acid β-oxidation and ameliorate insulin resistance (116, 117). Separate research also indicates a role for the STAT5/IGF-1 pathway in improving hepatic insulin resistance (118). Furthermore, APS addresses the low-grade inflammation associated with metabolic dysregulation. It attenuates the secretion of pro-inflammatory cytokines such as IL-6, an effect mediated through direct action on metabolic cells, which indirectly contributes to improved insulin sensitivity (119).

4.2. Effects against non-alcoholic fatty liver disease

Non-alcoholic fatty liver disease (NAFLD) is a chronic hepatic disorder intimately linked to metabolic syndrome, with a spectrum ranging from simple steatosis to non-alcoholic steatohepatitis (NASH), which can progress to fibrosis, cirrhosis, and hepatocellular carcinoma. Driven by global pandemics of obesity and type 2 diabetes, NAFLD affects roughly one-quarter of adults worldwide (120). Critically, there are no FDA-approved pharmacotherapies for NAFLD, making the development of safe, effective treatments a pressing unmet need (121). Consequently, the development of safe and effective treatments for NAFLD represents a pressing unmet need in medical research.

The mechanisms of APS against NAFLD highlight its pleiotropic, multi-target effects beyond immunomodulation. APS directly targets key pathological processes in hepatocytes, including lipid accumulation, oxidative stress, and fibrosis progression. Furthermore, APS alleviates hepatic steatosis through several interconnected pathways. It facilitates the decomposition of intracellular lipid droplets by regulating the SIRT1-FoxO1-mediated autophagy axis (122). Additionally, APS mitigates hepatic glucolipid metabolic disturbances induced by stress through activation of the SIRT1/AMPK/PPARα/PPARγ signaling cascade. Concurrently, APS modulates crucial signaling pathways responsible for lipogenesis, including mTOR/4EBP1/S6K1, thereby suppressing hepatic de novo lipogenesis. Endoplasmic reticulum stress is a major contributor to hepatocellular injury during NAFLD progression. Additionally, APS protects against hepatocellular injury by attenuating endoplasmic reticulum stress, as evidenced by the inhibition of the GRP78/IRE-1/JNK pathway, which shields hepatocytes from apoptotic death and decelerates NAFLD advancement (117).

4.3. Direct antitumor effects

While considerable focus has been placed on the immunoadjuvant role of APS in oncology (11, 123), extensive in vitro evidence confirms that APS also exerts direct anti-neoplastic effects on cancer cells, manifesting as the inhibition of proliferation, induction of apoptosis, and suppression of metastatic potential (124, 125). These direct effects are mediated through multiple mechanisms. A primary effect is the induction of apoptosis and cell cycle arrest. APS promotes tumor cell apoptosis by modulating diverse signaling pathways, such as suppressing the Wnt/β-catenin pathway or altering the expression balance of p53 and Bcl-2/Bax proteins to trigger the caspase cascade (70, 126). Concurrently, APS suppresses aberrant proliferation by targeting cell cycle regulators like CCNB1 and CDC6, inducing arrest at the G0/G1 or G2/M checkpoints (127). APS potently inhibits the proliferation of carcinoma cells derived from the breast, lung, and liver (125, 128, 129). The underlying mechanisms involve the suppression of pivotal oncogenic signaling pathways, including EGFR, Notch, and JAK/STAT3 that drive proliferation and metastasis (130, 131).

Furthermore, APS effectively attenuates cancer metastasis by impeding the epithelial-mesenchymal transition (EMT) program and reducing the activity of matrix metalloproteinases (MMPs), thereby limiting invasive potential (126). A significant translational application of APS is its ability to potentiate chemosensitivity. Acting as a chemopotentiating agent, APS demonstrates marked synergistic anti-tumor activity when co-administered with conventional chemotherapeutics such as cisplatin and doxorubicin (65, 132). Proposed mechanisms encompass the modulation of drug efflux pumps (e.g., P-glycoprotein) to elevate intracellular drug levels (133); the regulation of autophagic flux to overcome chemoresistance (134), and the alteration of specific microRNA expression profiles (e.g., miR-195-5p) to re-sensitize cells to therapy (135).

4.4. Anti-inflammatory effects

Inflammation represents a fundamental defensive response of the innate immune system to insult or injury and is intimately linked to the pathogenesis of a wide spectrum of diseases. However, uncontrolled or chronic inflammation can be profoundly detrimental to health, contributing to the development of conditions such as inflammatory bowel disease, arthritis, neurodegenerative disorders, and cardiovascular disease. APS modulates inflammatory signaling via multi-target mechanisms, positioning it as a significant candidate for the development of natural anti-inflammatory therapeutics. In both LPS-stimulated macrophage cultures and in vivo animal models, APS markedly suppresses the release of key pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-6, resulting in attenuated tissue inflammation and injury (136). These observations provide direct experimental support for the potential of APS as an effective anti-inflammatory agent. NF-κB is often termed the “master regulator” of inflammation, and its activation is a pivotal event initiating the transcription of a vast array of pro-inflammatory genes. Several studies (137, 138) have demonstrated that APS inhibits NF-κB activation by impeding the phosphorylation and proteasomal degradation of its inhibitory protein, IκBα. This results in the cytoplasmic retention of NF-κB, preventing its nuclear translocation and the subsequent initiation of pro-inflammatory gene transcription. Studies also report that APS can inhibit the phosphorylation of p38, ERK, and JNK, thereby blocking the transmission of upstream signals to nuclear transcription factors and cooperatively suppressing the inflammatory response. Furthermore, APS exerts its anti-inflammatory effects by inhibiting the JAK-STAT and PI3K/Akt signaling pathways. Oxidative stress can lead to an increase in intracellular oxygen free radicals and other reactive oxygen species, which can activate inflammatory responses, causing the infiltration of inflammatory cells and the release of inflammatory mediators. Research has found (139) that APS can activate nuclear factor erythroid 2-related factor 2 (Nrf2), promote its nuclear translocation, and induce the expression of downstream antioxidant proteins such as heme oxygenase-1 (HO-1), thereby scavenging reactive oxygen species (ROS), alleviating oxidative stress, and indirectly achieving anti-inflammatory effects through antioxidant actions.

Furthermore, studies have found that APS exhibits crosstalk with energy metabolism pathways. For example, the anti-inflammatory effects induced by APS in palmitate-treated RAW264.7 cells are dependent on the activity of AMP-activated protein kinase (AMPK), revealing that APS may influence the inflammatory process by modulating cellular energy metabolic status (10). The gut microbiota is regarded as the body’s “second genome,” and its homeostasis is crucial for host immune function. The anti-inflammatory effects of orally administered APS are largely achieved by remodeling the structure of the gut microbiota (140). Functioning as a prebiotic, APS can promote the growth of Lactobacillus and Bifidobacterium, inhibit harmful bacteria, and modulate microbial metabolites (e.g., short-chain fatty acids), thereby restoring intestinal barrier function, modulating systemic immune responses, and ultimately reducing inflammation in distal organs (141, 142). This regulatory mechanism involving the “gut-brain axis” and “gut-lung axis” opens new perspectives for the application of APS.

4.5. Other activity

Mao et al. (143) discovered that Astragalus polysaccharide and its congener astragaloside IV significantly inhibit the expression of TGF-β1 and the phosphorylation of its downstream Smad2/3 proteins, thereby blocking the activation of myofibroblasts, reducing the excessive production and deposition of extracellular matrix components such as collagen and fibronectin, and consequently exerting an anti-hepatic fibrotic effect. Furthermore, Astragalus polysaccharide exhibits protective effects in various models of neurological damage. For example, in a diabetic rat model (144), APS was able to ameliorate learning and memory impairments and reduce neuronal death, a mechanism potentially related to the upregulation of phosphorylation levels of CREB, NMDA receptors, and CaMK II.

5. Cutting-edge applications and delivery innovations

5.1. The combination of Astragalus polysaccharide

The strategic combination of APS with conventional therapies represents a prominent trend, driven by the goals of achieving synergistic efficacy, attenuating side effects, and overcoming drug resistance. A key application is the enhancement of chemotherapy. Clinical research indicates that intravenous APS, when co-administered with paclitaxel and cisplatin, is applicable for treating advanced non-small cell lung cancer (145). A meta-analysis further confirmed that APS injection combined with the FOLFOX regimen significantly enhances therapeutic outcomes and mitigates adverse events in gastric cancer patients (146). Beyond clinical observations, the mechanistic basis for this synergy has been elucidated. For instance, APS potentiates the effect of cisplatin in ovarian cancer cells by activating the JNK pathway, which in turn regulates key apoptosis proteins like Bcl-2, Bax, and Caspase-3 (147). Furthermore, APS can counteract multidrug resistance MDR, a major cause of chemotherapeutic failure. Evidence suggests that APS re-sensitizes tumor cells by suppressing the expression of efflux pumps like P-glycoprotein, thereby increasing intracellular drug retention (148), In addition to enhancing efficacy, APS plays a critical role in mitigating chemotherapy-induced toxicity. Robust evidence from meta-analyses demonstrates that Astragalus-containing preparations significantly lower the risk of severe toxicities, including neutropenia, anemia, and gastrointestinal events (149–151). These systemic benefits are complemented by organ-specific protective effects. Research indicates that APS effectively attenuates anthracycline-induced cardiotoxicity, as evidenced by better preservation of cardiac function in patients (152, 153). The combinatory potential of APS extends beyond oncology into metabolic and inflammatory diseases. For example, a combination of Malus micromalus polysaccharide and APS ameliorated alcoholic fatty liver disease via modulation of the gut microbiota, while APS and matrine acted synergistically against ulcerative colitis (154, 155). Collectively, this evidence highlights the considerable potential of APS as a versatile “platform” therapeutic, capable of being integrated into multi-agent regimens to enhance efficacy and reduce toxicity across a spectrum of complex diseases.

5.2. Applications as a potential natural carrier material

Nanoparticle delivery systems have become a central focus in APS delivery research due to their advantages in protecting the drug, improving solubility, prolonging in vivo circulation time, and enabling targeted delivery. Poly (lactic-co-glycolic acid) (PLGA) is an FDA-approved biodegradable and biocompatible polymer widely used for drug delivery (156, 157). Researchers have successfully prepared PLGA nanoparticles loaded with APS (158). These nanoparticles can not only effectively encapsulate APS but also act as an immune adjuvant to enhance its immunogenicity (159). One study co-encapsulated APS and gold nanorods (AuNRs) into PLGA-polyethylene glycol (PEG) nanoparticles for combined photothermal-immunotherapy of breast cancer. The results showed that the encapsulation efficiency (EE) for APS could reach 54.89 ± 2.07% (160). Following local injection into the tumor, these nanoparticles effectively accumulated in the tumor tissue, exerting a synergistic therapeutic effect.

Liposomes, as a classic nanocarrier, are highly suitable for delivering polysaccharide-based drugs due to their cell membrane-like structure and excellent biocompatibility (161). Studies have shown that formulating APS into Astragalus polysaccharide liposomes (APS-Ls) can significantly improve its stability and delivery efficiency. One study successfully prepared APS-Ls with a particle size of less than 150 nm, achieving an entrapment rate of 47.50% ± 0.15% and a drug loading capacity of 5.40% ± 0.17% (162). Liposomes can protect APS from degradation by endogenous enzymes, prolong its circulation time, and potentially amplify its immunomodulatory effects by enhancing cellular uptake. Encapsulation efficiency (EE) and loading capacity (LC) are key metrics for evaluating the drug loading capability of nanocarriers. The EE of APS in PLGA and liposome systems can reach approximately 55% and 48%, respectively.

Employing APS as a self-assembling nanocarrier constitutes a remarkably innovative and fascinating recent research avenue. Research has found that APS molecules themselves possess the ability to self-assemble into nanoparticles (163). APS molecular chains contain numerous hydrophilic groups; however, under specific conditions, they can spontaneously aggregate via weak intermolecular interactions such as hydrogen bonding and van der Waals forces, forming nano-sized aggregates with internal hydrophobic cavities. This intrinsic property positions APS not merely as a bioactive drug substance, but also as a natural nanocarrier with exceptional biocompatibility (164).

5.3. Vaccine adjuvant

Vaccines represent the most effective public health tool for the prevention and control of infectious diseases. However, many modern vaccines, particularly subunit vaccines, recombinant protein vaccines, and nucleic acid vaccines, possess inherently weak immunogenicity and require the addition of an adjuvant to induce a sufficiently robust and durable protective immune response (165, 166). Adjuvants enhance, modulate, and prolong the host’s immune response to the antigen, thereby improving vaccine efficacy (167). Extensive research indicates that APS, when used as an adjuvant, can significantly enhance the immunogenicity of vaccines. For example, in various vaccine models (e.g., against H5N1 avian influenza, Newcastle disease virus, or ovalbumin), the addition of APS as an adjuvant consistently significantly elevated the titers of specific antibodies, including total IgG and key protective antibodies such as hemagglutination inhibition (HAI) antibodies (168–170). This indicates that APS can effectively promote the activation, proliferation, and differentiation of B cells into plasma cells, leading to abundant antibody production. Furthermore, APS can significantly promote the proliferative response of T lymphocytes, particularly splenocytes. In an influenza vaccine model, sera from the APS-adjuvanted group displayed elevated levels of both Th1 (e.g., IFN-γ, TNF-α) and Th2 (e.g., IL-4) cytokines, indicative of its capacity to elicit a balanced Th1/Th2 immune response. Comparative analyses revealed that APS adjuvantation was superior to either the vaccine alone or a standard aluminum adjuvant in enhancing HAI antibody titers and IgG levels in mice, and it significantly increased survival rates following viral infection. Notably, a low dosage of APS adjuvant demonstrated efficacy marginally superior to that of aluminum adjuvant, while concurrently inducing a more tempered inflammatory reaction (171). Collectively, these preclinical findings provide robust evidence endorsing APS as a potent and versatile vaccine adjuvant.

Mechanistic studies indicate that APS, via activation of the TLR4 signaling pathway, induces the secretion of Th1-polarizing cytokines (e.g., IL-12) alongside Th2-type cytokines (e.g., IL-4), facilitating a balanced Th1/Th2 hybrid immune response (172). This capacity for “bidirectional immunomodulation” constitutes a fundamental and distinguishing advantage of APS (173, 174).

Yakuboğulları et al. (175)utilized Astragalus polysaccharide (APS, gum tragacanth) and Astragaloside VII (AST-VII) to construct two nano-adjuvant carrier systems (APNS and ANS) and investigated their immunoenhancing effects as adjuvants for a seasonal influenza A (H3N2) vaccine. The study prepared nanoparticles via a pH-driven self-assembly method and evaluated the induced humoral immunity, cellular immune responses, and protective efficacy in a mouse model. The experimental results demonstrated that the APS-based system (APNS) primarily induced a Th2-biased immune response, characterized by a significant increase in vaccine-specific IgG1 antibody titers. In contrast, the composite nanoparticle integrating AST-VII with APS (ANS) guided a balanced Th1/Th2 immune response with mild Th17 activation, evidenced by the concurrent elevation of both IgG1 and IgG2a antibodies, promotion of IFN-γ and IL-17A secretion, and a remarkable enhancement of splenocyte proliferation. The underlying mechanism involves: APS, acting as both a carrier and immunomodulator, primarily drives the Th2 response by promoting B-cell activation and antibody production. Meanwhile, AST-VII, a triterpenoid saponin, synergizes with APS to balance T-cell differentiation, promoting the secretion of Th1-type (e.g., IFN-γ) and Th17-type (e.g., IL-17A) cytokines, thereby collectively shaping a balanced immune profile that combines strong antibody responses with robust cellular immunity.

Additionally, Yakuboğulları et al. (175, 176) systematically evaluated the immunoenhancing effects of Astragalus polysaccharide (APS) and Astragaloside VII (AST-VII) as nano-adjuvant carriers for both seasonal influenza A (H3N2) and Newcastle Disease Virus (NDV) vaccines. In these studies, nanoparticles were prepared via a pH-driven self-assembly method and administered to mouse models to assess humoral immunity, cellular immune responses, and protective efficacy. The results revealed distinct yet complementary roles for APS and AST-VII. The APS-based system (APNS) primarily induced a Th2-biased immune response, marked by a significant increase in vaccine-specific IgG1 antibody titers. In contrast, the composite nanoparticle integrating AST-VII with APS (ANS) guided a balanced Th1/Th2 immune response with mild Th17 activation, evidenced by the concurrent elevation of IgG1 and IgG2a antibodies, promotion of IFN-γ and IL-17A secretion, and enhanced splenocyte proliferation. Notably, in the NDV vaccine model, APS-based systems significantly boosted neutralizing antibody titers, outperforming a commercial oil-adjuvanted vaccine. Mechanistically, APS functions dually as both a delivery vehicle and an immunomodulator, primarily driving potent humoral immunity by enhancing B-cell responses. Meanwhile, AST-VII, a triterpenoid saponin, orchestrates T-cell responses by promoting balanced secretion of Th1- and Th2-related factors, thereby shaping an immune profile that effectively combines robust antibody production with cellular immunity.

This property enables APS-adjuvanted vaccines to stimulate not only high-titer neutralizing antibodies but also robust cytotoxic T lymphocyte (CTL) responses, offering a “dual-layered” protective strategy against challenging pathogens. A significant challenge, however, stems from the inherent nature of APS as a heterogeneous botanical extract; its chemical composition, molecular weight distribution, and purity are subject to variation based on the geographical source of the raw material and the specific extraction protocols employed, factors that directly influence its biological activity and safety profile. Therefore, the establishment of standardized manufacturing processes and rigorous quality control metrics is an essential prerequisite for the successful development of APS as a pharmaceutical-grade adjuvant. While TLR4 activation is implicated in its mechanism, the specific molecular entities within the complex APS mixture that are responsible for its core adjuvant activity, along with the complete intricate mechanism of its immunoregulatory network modulation, require further in-depth elucidation. Moreover, employing advanced techniques like systems biology and multi-omics approaches to comprehensively decipher its mode of action will be crucial for optimizing its application and potentially identifying novel biomarkers.

5.4. Applications in animal husbandry

Within the global context of pursuing green and sustainable development, the agriculture and animal husbandry sectors are experiencing a significant transformation. The escalating problem of antimicrobial resistance (AMR), driven by the misuse of antibiotics in animal feed, has prompted nations worldwide to implement policies restricting or banning their use as growth promoters. Consequently, identifying safe and efficacious antibiotic alternatives has emerged as a critical industry priority and an urgent necessity (23, 177). In light of increasingly stringent global regulations on in-feed antibiotics, the value of APS as a promising natural alternative has gained significant prominence (178). Studies (179) demonstrate that dietary supplementation with APS in cyclophosphamide (CPM)-treated broiler chickens enhances immune organ indices (e.g., spleen, thymus), stimulates immune cell proliferation and plasma nitric oxide synthase (NOS) activity, and significantly increases serum levels of immunoglobulins (IgG, IgA, and IgM). The gastrointestinal tract serves as the primary defense barrier for animal health. APS has been demonstrated to effectively ameliorate intestinal health (180). Its beneficial effects are multi-faceted: 1) APS optimizes gut morphology by increasing villus height and crypt depth, thereby expanding the absorptive surface area and enhancing the digestibility and absorption of nutrients; 2) It modulates the gut microbial community structure by inhibiting the growth of pathogenic bacteria and promoting the proliferation of beneficial bacteria (e.g., Lactobacillus and Bifidobacterium), thus maintaining a healthy microbial equilibrium; 3) It strengthens intestinal barrier integrity, reduces the absorption of endotoxins (e.g., LPS), and attenuates inflammatory responses (23, 181, 182).

6. Challenges and future perspectives

Despite notable advancements, the clinical translation of Astragalus polysaccharide (APS) is hindered by several interconnected challenges that define a clear roadmap for future research. These challenges can be grouped into two primary categories: the fundamental need for standardization and mechanistic validation, and the practical requirements for clinical translation.

The first category encompasses the need to overcome structural heterogeneity and establish causal mechanisms. A primary obstacle is the lack of standardization; future work must define critical quality attributes (e.g., molecular weight distribution, monosaccharide ratios) and establish a minimum data package for preclinical and clinical batches. To bridge the gap between structure and function, a prioritized research agenda should focus on: (1) validating direct receptor interactions using techniques like SPR/ITC with purified fractions and receptor ectodomains (e.g., TLR4/MD-2, MR); and (2) employing standardized functional assays in receptor-knockout models or with specific inhibitors to unequivocally attribute immunomodulatory effects. Underpinning these goals is the fundamental challenge of structural elucidation, which requires the application of advanced spectroscopy and AI to decipher structure-activity relationships.

The second category involves the translation of this mechanistic understanding into viable therapies. This requires a sophisticated PK/PD strategy using labeled fractions to track biodistribution and correlate structural features with exposure and immune readouts. From a manufacturing perspective, overcoming source variability is critical; this will likely involve synthetic biology and chemoenzymatic approaches to produce defined oligosaccharides, alongside navigating regulatory pathways for complex mixtures. Finally, clinical translation demands innovative trial designs with defined endpoints and biomarkers (e.g., cytokine panels, DC activation markers) for specific patient populations, such as in vaccine adjuvancy or chemo-immunotherapy.

Looking forward, research is poised to advance on the principles of precision and standardization. The integration of technologies like single-cell sequencing and spatial transcriptomics will provide a deeper, systems-level understanding of APS’s mechanisms. It is anticipated that through these concerted efforts, APS will evolve beyond its traditional origins into a novel class of glyco-pharmaceuticals with well-defined structures, playing a significant role in immunotherapy, chronic disease management, and the “One Health” paradigm.

7. Conclusion

Modern pharmacological investigations have uncovered the material basis for the diverse biological activities of Astragalus (Radix Astragali), a primary herb used for thousands of years in Traditional Chinese Medicine (TCM) to “tonify Qi.” Among its constituents, APS is widely acknowledged as one of its most significant and plentiful bioactive compounds. APS is not a single chemical entity but rather a heterogeneous class of complex biopolymers consisting of multiple monosaccharides connected via diverse glycosidic linkages. Our study provides a systematic dissection of the structural heterogeneity of APS and its immunoregulatory network, which operates as a multi-layered system with TLR4/MyD88 as its core, MR/NOD receptors as co-regulators, and T cell reprogramming as the effector mechanism. Through the application of multi-dimensional analytical techniques, we have elucidated the quantitative structure-activity relationship governing the interplay between its chemical structure, conformational dynamics, and receptor engagement. We have also systematically characterized the pleiotropic activities of APS extending beyond immunomodulation, positioning it as a “polyfunctional molecular weapon” with therapeutic potential against cancer, metabolic disorders, and inflammatory diseases.

Funding Statement

The author(s) declare financial support was received for the research and/or publication of this article. This work was supported by the Natural Science Foundation of Ningxia(2021AACC03299), Key Research and Development Projects of Ningxia Hui Autonomous Region(2022BEG03123), 2024 Program for Cultivating Outstanding Young Talents in Ningxia Hui Autonomous Region, and Science and Technology Innovation Project of Yinchuan City(2024SF005).

Author contributions

BW: Data curation, Formal Analysis, Investigation, Writing – original draft, Writing – review & editing. BLW: Data curation, Formal Analysis, Writing – original draft, Writing – review & editing. YM: Data curation, Formal Analysis, Writing – original draft, Writing – review & editing. XL: Data curation, Formal Analysis, Resources, Software, Writing – original draft, Writing – review & editing. LT: Data curation, Formal Analysis, Resources, Writing – original draft, Writing – review & editing. LJ: Data curation, Formal Analysis, Resources, Writing – original draft, Writing – review & editing. XZ: Funding acquisition, Project administration, Resources, Writing – original draft, Writing – review & editing. XD: Project administration, Resources, Supervision, Writing – original draft, Writing – review & editing.

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Generative AI statement

The author(s) declare that no Generative AI was used in the creation of this manuscript.

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