A. membranaceous, a common qi-tonifying herb, has had its medicinal use documented since 'Shen Nong's Herbal Classic,' written over 2,000 years ago in China (28). It is a member of the legume family and has a wide geographical distribution across the Northern Hemisphere, South America and Africa, featuring a distinctive plant morphology with erect and well-branched stems and neatly arranged leaflets on pinnately compound leaves (29). AS-IV is a natural saponin extracted from the root of A. membranaceous, also known as RA. The composition of RA varies by region and country. Its main components include astragali polysaccharides, saponins and flavonoids (25). AS-IV serves as a marker substance for one of the aspects of quality control for RA and its products in the Chinese Pharmacopoeia (2020 edition) (30).

RA is used in the field of ethnopharmacology in several countries in Asia, North America and Europe, such as China, the United States and Russia (31). In clinical practice, numerous proprietary and patented Chinese medicines and injections are formulated using or contain RA, including Astragalus injection, Astragalus polysaccharide injection, Yupingfeng Powder, Xuefu Zhuyu Decoction and Huangqi Siwu Decoction (32,33). These preparations have shown favorable application prospects in treating neurological, cardiovascular, renal, gynecological and pulmonary disorders, and organ injuries (26). RA is used as a dietary product in China and is also classified as a legal dietary supplement in the United States (34).

AS-IV appears as a white to yellow crystalline powder (35) and has a chemical name of 3-O-β-d-xylopyranosyl-6-O-β-glucopyranosyl-cyclostelli feryl alcohol, molecular formula of C₄₁H₆₈O_14, and a relative molecular mass of 784.97 g/mol (36). This compound has a high melting point, ranging from 295-296°C. It is insoluble in water but displays solubility in certain organic solvents such as ethanol, methanol and acetone (37). AS-IV belongs to the category of triterpenoid saponins and pentacyclic triterpenoids, featuring a tetracyclic triterpenoid saponin in the form of lanolin ester alcohol. This structural configuration is vital because it underlies the manifestation of its anti-inflammatory and antioxidant effects and ability to improve lipid metabolism, thereby potentially exerting hepatoprotective effects (38,39). The commonly employed extraction methods include solvent extraction, ultrasound-assisted extraction, microwave-assisted extraction, enzyme-assisted extraction, supercritical fluid extraction and rapid extraction techniques (40) [Fig. 1 (37)].

AS-IV, an emerging natural compound in the medical realm, is a highly promising nutritional therapeutic agent. It displays an array of advantageous biological functions, such as anti-inflammatory, antioxidant (41), antiapoptotic (42), anticancer (43) and antitumor (44). These properties hold promise for its applications in neurological disorders such as stroke, dementia, epilepsy and Alzheimer's disease (29). AS-IV is also of great significance in cardioprotection. It may play a role, either directly or indirectly, in the treatment of cardiac disorders. It controls myocardial hypertrophy and fibrosis processes and effectively reduces cardiomyocyte apoptosis (45-47). In addition, AS-IV shows remarkable efficacy in combating diabetes and its complications (48), along with certain viruses (49). In the area of intestinal health, it modulates intestinal function, alleviates intestinal inflammation, and effectively restores damaged intestinal barrier function (50,51). In the treatment of kidney and gynecological diseases, AS-IV can ameliorate chronic glomerulonephritis and triple-negative breast cancer by participating in autophagy inhibition (52,53). Owing to its powerful antioxidant and anti-inflammatory characteristics, AS-IV exhibits significant protective effects against various lung diseases (54,55). These properties provide a strong rationale for the evaluation of AS-IV as a potential therapeutic option for various liver ailments.

NAFLD belongs to the category of metabolic syndromes. It is triggered by well-defined liver-damaging factors. A prominent feature of NAFLD is the over-accumulation of fat in hepatocytes, which is evident in conditions such as non-alcoholic fatty liver, non-alcoholic steatohepatitis (NASH) and associated LC (56). In general, non-alcoholic fatty liver may progress to NASH, which in turn may eventually progress to LC and liver cancer (57). NAFLD development is associated with a variety of factors. The currently widely recognized 'multiple strike' hypothesis takes into account the synergistic effects of fat build-up, lipotoxicity, inflammation, oxidative stress, mitochondrial impairment, gut microbiota and genetic elements (58). As shown in NAFLD models, a diet rich in fat can trigger hepatic steatosis and inflammation, which subsequently lead to significant oxidative damage in the liver (59). Palmitic acid, the most prevalent free fatty acid (FFA) in dietary sources and the serum, contributes to hepatic steatosis and exacerbates insulin resistance (60).

Zhai et al (61) successfully established a cellular model of hepatic steatosis using FFA. They discovered that FFA-induced lipid accumulation led to endoplasmic reticulum (ER) stress, and triggered autophagy and inflammation. This ER stress was significantly alleviated by AS-IV treatment. It reduced the elevated expression of ER stress markers, such as glucose-regulated protein 78, protein kinase R-like ER kinase, and C/EBP homologous protein, and mitigated the increased nuclear translocation of these markers (21,62). Liu et al (63) discovered that FFA induced the blockage of autophagic fluxes, attenuating lipophagy. AS-IV triggered autophagy within HepG2 cells by inhibiting the activation of protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway, effectively attenuating FFA-induced lipid accumulation in HepG2 cells and degrading excess intracellular lipids. Moreover, AS-IV alleviates oxidative stress in hepatocytes. It reduces the elevated levels of reactive oxygen species (ROS) and malondialdehyde (MDA) and mitigates the significant decrease in GSH peroxidase (Px) activity. And, AS-IV plays a role in hepatocyte apoptosis inhibition by downregulating the mRNA and protein expression levels of B-cell lymphoma/leukemia-2-associated X protein (Bax) and upregulating the mRNA and protein expression levels of B-cell lymphoma 2 (Bcl-2) (64). NAFLD is marked by the deposition of hepatic fat within the liver, and prolonged deposition can give rise to hepatic inflammation. AS-IV can mitigate the inflammatory response by inhibiting the generation of inflammatory cytokines such as interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). Toll-like receptor 4 (TLR4) can initiate a series of injury-related immune responses. However, the overexpression of the TLR4 signaling pathway leads to the occurrence of immune-inflammatory responses, and AS-IV can inhibit the overexpression of this pathway (64-67). AMPK functions as a crucial regulator in hepatic lipogenesis, and acetyl-CoA carboxylase phosphorylation is a direct target of AMPK. AS-IV dose-dependently increased the phosphorylation of acetyl-CoA carboxylase (ACC) by AMPK activation. The presence of adipogenic genes such as acc1, scd1 and fas in sterol regulatory element-binding protein-1c (SREBP-1c) reduces the accumulation of mature SREBP-1c in HepG2 cells exposed to FFA. It also inhibits the enhanced nuclear translocation of SREBP-1c and downregulates adipogenic gene mRNA. Co-processing with 5-amino-4-imidazolecarboxamide ribonucleotide can further enhance this effect (68). The intestinal flora is essential for maintaining physiological balance in the human body (69). When the population of bile salt hydrolase flora decreases, AS-IV can inhibit diet-induced metabolic damage, especially hepatic steatosis. AS-IV can also inhibit intestinal farnesoid receptor (FXR) signaling, and this inhibition is accompanied with a reduction in intestinal fibroblast growth factor expression. Subsequently, hepatic FXR is activated. AS-IV increases glucagon-like peptide-1 and promotes ceramide production. All these effects collaborate to suppress the progression of hepatic steatosis (61). In a mouse model study of NAFLD, AS-IV reduced body weight gain in NAFLD mice without reducing food intake. It also decreased the levels of NAFLD markers aspartate aminotransferase (AST) and alanine aminotransferase (ALT), eliminated the tendency of elevated triglyceride (TG) and total cholesterol, and significantly reduced the release of aminotransferases due to hepatocellular injury (64,70).

All the aforementioned molecular effects of AS-IV have been validated across heterogeneous experimental systems, including in vitro fatty liver cell models, HepG2 cell lines, primary mouse hepatocytes, and in vivo NAFLD mouse and rat models. Comprehensive analysis of current evidence demonstrates that, despite variations in experimental conditions, these regulatory effects maintain an overall consistent trend. AS-IV directly reduces fat accumulation, facilitates excess fat degradation, and inhibits steatosis by regulating autophagy, promoting AMPK activation, and synergizing with the gut microbiota. Furthermore, it suppresses oxidative stress and hepatic inflammation in hepatocytes, thereby alleviating NAFLD.

LF is the outcome of a continuous wound-healing response to chronic liver injury, and its main causes include chronic hepatitis C virus (HCV) and hepatitis B virus (HBV) infections, alcohol abuse that results in alcoholic liver disease (ALD), NASH, and autoimmune diseases (71). The distinguishing feature of LF is HSC activation, which subsequently triggers excessive ECM accumulation, leading to fibrous scarring. If the disease progresses further, fibrous scarring can transform into LC and ESLD. LC is defined as the presence of regenerating hepatocellular nodules in the liver, accompanied with a reduced hepatic blood supply. Oxidative stress and inflammation are as important factors in LF development (72,73).

Chen et al (74) revealed the protective effects of AS-IV on LF, it downregulated collagen type I alpha 1 chain, α-smooth muscle actin (α-SMA) and FN, thereby reducing the excessive secretion and accumulation of ECM. AS-IV can also inhibit the excessive pathological activation of the NF-κB signaling pathway, thereby exerting antifibrotic effects. It can enhance the expression levels of p65, p52 and p50, thus promoting the senescence and apoptosis of activated HSC-T6 cells. Moreover, AS-IV reduces the protein and mRNA expression of α-SMA, collagen synthesis, and FN through mediated p38 MAPK. In turn, this process inhibits HSC activation. AS-IV exhibits antioxidant properties and could decrease the levels of ROS and lipid peroxide (75). Hydroxyproline content, one of the indicators of LF, initially showed a decreasing trend after AS-IV treatment. In vitro studies showed that the platelet-derived growth factor-bb stimulated proliferation of HSC was significantly inhibited by AS-IV (76). In the bile duct ligation-induced LF model, the combined treatment of AS-IV and ferulic acid (FA) effectively alleviated LF, significantly reduced collagen deposition, and greatly improved the pathological changes in the liver. Compared with AS-IV or FA alone, the combination therapy was more effective in inhibiting HSC activation and significantly enhanced the body's capacity to resist oxidative stress. The reduction in MDA levels, elevation in GSH-Px, and enhancement of superoxide dismutase (SOD) enzyme activity are all attributed to AS-IV rather than FA. In addition, AS-IV activates antioxidant defense mechanisms via the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway and promotes the phosphorylation of glycogen synthase kinase 3β (GSK-3β) at the Ser9 site (77).

All the aforementioned molecular effects of AS-IV have been validated across heterogeneous experimental systems, including in vitro HSC-T6 cells and HCC cell lines, in vivo models of the PS-induced LF rat model, the bile duct ligation rat model, and the PSmad3C and Nrf2 mouse models. Comprehensive analysis of current evidence demonstrates that, despite variations in experimental conditions, these regulatory effects maintain an overall consistent trend. AS-IV exerts antifibrotic effects in vivo by inhibiting oxidative stress and inflammation via the NF-κB and Nrf2 pathways. It also suppresses the proliferation of HSC in vitro, and its combined treatment with FA synergistically enhances the anti-fibrotic effect. Compared with other antiviral drugs, corticosteroids, immunosuppressive and antifibrotic agents, AS-IV exhibits significant advantages, including a broad application spectrum, high safety, multi-target regulation and low cost (78,79) (Fig. 2).

Liver cancer, a common solid malignancy arising from the liver, is the sixth most common type of cancer and the fourth leading cause of cancer-related deaths worldwide (80). It encompasses HCC, intrahepatic cholangiocarcinoma and fibrous laminar carcinoma. Among them, HCC is the most common type of primary liver cancer, accounting for 90% of all liver cancer cases (81-83). Numerous factors, including HBV and HCV infections (84), NAFLD (85), alcohol abuse (86) and exposure to aflatoxin (87,88), elevate the risk of developing HCC. The common symptoms of HCC are abdominal pain, abdominal distension, weight loss, fatigue, jaundice and vomiting (89). Its pathogenesis is intricate and may involve alterations in the tumor microenvironment, inflammatory response, oxidative stress, tissue hypoxia, hepatocyte necrosis, regeneration related to fibrosis, and numerous molecular-level changes (90,91). At present, surgical resection remains the preferred treatment for HCC (92). AS-IV, a typical natural antioxidant, exerts potential pharmacological effects against various malignancies. It influences the development of HCC via four principal mechanisms: (i) metabolic regulation, (ii) immunomodulatory mechanism, (iii) apoptosis induction, and (iv) inhibition of epithelial-mesenchymal transition (EMT). The specific mechanisms are described below:

First, AS-IV can inhibit the malignant proliferation of hepatoma cells by regulating glycolipid metabolism, thereby breaking the abnormal metabolic homeostasis of tumor cells. A recent study showed that AS-IV decreased glucose consumption, lactate production and extracellular acidification rate (93). It also inhibits phosphoglycerate mutase 1 (PGAM1) succinylation levels and lysine acetyltransferase 2A (KAT2A) protein levels in SNU182 and Huh7 cell lines. Overexpressing KAT2A counteracted the reduction in cell viability and glycolysis brought about by AS-IV treatment in the SNU182 and Huh-7 cell lines. Meanwhile, KAT2A increases the succinylation of PGAM1, and AS-IV appeared to inhibit tumor growth by blocking the KAT2A-mediated succinylation of PGAM1 (93). Fang Gong et al (94) and Li et al (95) revealed that AS-IV significantly reduces the proliferation, migration and invasion in Huh-7 cells and suppresses ROS production. These effects were achieved via multiple pathways, including the TGF β1/mothers against decapentaplegic homolog (Smad) and Nrf2/heme oxygenase 1 (HO-1) pathway; upregulated expression of pSmad3C, pNrf2, HO-1 and DT-diaphorase (NQO1); and decreased expression of pSmad2C, pSmad2L, pSmad3L, plasminogen activator inhibitor-1 and α-SMA. The inhibitory impact of AS-IV on HCC in vivo was further corroborated in a mouse subcutaneous tumor model, in which AS-IV suppressed the proliferation, migration and invasive capacity of Huh-7 cells by inhibiting macrophage M2 polarization and reducing the expression of M2 macrophage-stimulated phosphorylated signal transducer and activator of signal transducer and activator of transcription (STAT) 3, p-NF-κB and TLR4 expression (94,96). In a mouse model of HCC, Li et al (95) analyses further verified the reversibility and antagonism of AS-IV in regulating pSmad3C/3L and the phosphorylated expression of the Nrf2/HO-1 pathway in TGF β1-activated HSC-T6 and HepG2 cells. All these results indicated that AS-IV can effectively inhibit HCC progression by suppressing the proliferation, migration and invasion of liver cancer cells.

AS-IV exerts indirect anti-hepatoma effects by regulating the tumor immune microenvironment and enhancing antitumor immune responses. Yang et al (97) and Cui et al (98) uncovered the underlying mechanism: AS-IV could target the miR-135b-5p signaling pathway of carboxypeptidase n domain-containing protein 1, alleviating the immunosuppression caused by programmed death-ligand 1 (PD-L1) and accelerating apoptosis. In addition, AS-IV exhibited a dose-dependent inhibitory impact on the proliferation of Huh-7 and SMMC-7721 cell lines. It suppressed the interferon-γ-induced expression of PD-L1, thereby weakening PD-L1-mediated immunosuppression and immune cell cytotoxicity, and strengthening the antitumor immune response (96). Guo et al (99) demonstrated that AS-IV facilitated dendritic cell maturation and enhanced antigen presentation. It also increased the release of the functional cytokine interleukin-12 (IL-12) by upregulating various surface markers, namely, cluster of differentiation (CD)14, CD40, CD80, CD83, CD86 and human leukocyte antigen-DR. As a consequence, dendritic cell-induced specific cytotoxic T lymphocyte responses against the HCC cell line SMMC-7721 were enhanced. IL-12 is regarded as a natural immune booster in dendritic cell-mediated anti-HCC therapy. In a study by Qi et al (100), AS-IV exhibited anticancer activity by downregulating oncogenes such as vav guanine nucleotide exchange factor 3.1 and inhibiting the non-adherent-dependent growth of HepG2 cells. Another study showed that AS-IV inhibited the phosphorylation of c-Jun-terminal kinase and p-c-Jun and downregulated multidrug resistance gene 1 in Bel-7402 and Bel-7402/FU cells in a dose-dependent manner (101). It achieved this feature by decreasing the expression levels of multidrug resistance gene 1 mRNA and p-glycoprotein, thus reversing the resistance of Bel-7402/FU cells to 5-FU. Therefore, AS-IV can reverse multidrug resistance in a mouse model of HCC (101). Meanwhile, Qu et al (102) discovered that AS-IV enhanced the antitumor effect of cisplatin by inhibiting the expression of certain factors in HepG2 cells and multidrug resistance-associated protein 2. It also enhanced the responsiveness of HCC to cisplatin-based chemotherapy.

Inducing the apoptosis of hepatoma cells is one of the core antitumor mechanisms of AS-IV. Cui et al (98) revealed that AS-IV negatively regulated CTNNB1 levels through the binding of miR-150-5p to the untranslated region of CTNNB1. In addition, CTNNB1 overexpression partially reversed the tumor growth inhibition and apoptosis effects induced by miR-150-5p upregulation. Meanwhile, miR-150-5p upregulation suppressed β-catenin expression, thereby promoting apoptosis and inhibiting tumor growth. Furthermore, Su et al (103) showed that AS-IV could induce SK-Hep1 and Hep3B cells to halt the cell cycle in the G1 phase and activate the caspase-8-dependent exogenous and caspase-9-dependent endogenous apoptotic pathways. As a result, it suppressed the expression of apoptotic signal proteins such as myeloid cell leukemia-1 protein, x-linked inhibitor of apoptosis protein, cellular fas-associated death domain-like interleukin-1β-converting enzyme inhibitory protein, and survivin in HCC cells.

AS-IV significantly inhibits EMT in hepatoma cells. EMT is a key process for the migration, invasion and distant metastasis of hepatoma cells. Qin et al (104) reported that AS-IV could mitigate the inhibitory effect on GSK-3β by reducing Akt phosphorylation. As a consequence, the expression of the wingless-type MMTV integration site family (Wnt)/β-catenin signaling pathway is suppressed, and the EMT signaling pathway is regulated, resulting in the downregulation of N-catenin, vimentin and α-SMA. In addition, AS-IV reduces the expression of zinc finger protein transcription factor and promotes the transcription of E-cadherin to some extent, thereby weakening the invasion and migration abilities of HCC cells. Li et al (15) demonstrated that AS-IV inhibited the EMT and migration of HCC cells by downregulating long non-coding RNA-activated transcript in breast cancer. Jiang and Mao (105) demonstrated that AS-IV reduced the expression of N-calmodulin, E-calmodulin and vimentin in HepG2 and Hep3B cell lines by influencing their EMT. As a consequence, the proliferative capacity of these HCC cell lines HepG2 and Hep3B was diminished. Moreover, the enhanced apoptotic capacity was manifested by a substantial elevation in the protein amounts of caspase-3 and caspase-9, effectively inhibited tumor invasion and migration. In a mouse model of HCC, AS-IV induced the apoptosis of tumor cells by blocking the activation of the Wnt/β catenin/T cell factor-4 signaling pathway and reducing the protein expression of vascular endothelial growth factor.

All the aforementioned molecular effects of AS-IV have been validated across heterogeneous experimental systems, including in vitro HCC cell lines, in vivo nude mouse xenograft models, H22 tumor-bearing mouse models, HepG2 xenograft tumor models, and HCC rat models. Comprehensive analysis of current evidence demonstrates that, despite variations in experimental conditions, these regulatory effects maintain an overall consistent trend. AS-IV exerts a pivotal function in HCC development. It curbs the proliferation, migration, and invasion of cancer cells; restrains tumor growth; promotes apoptosis; and improves the treatment outcome by enhancing the antitumor immune response (Fig. 3).

Liver injury refers to a series of pathophysiological alterations induced by diverse internal and external factors, with significant manifestations of liver dysfunction (106). If this condition remains inadequately controlled, then it has a high probability of further progressing to LF or LC, and may even deteriorate into acute liver failure (107). Liver injury has numerous causes, including toxins, inflammation, metabolic disorders and ischemia-reperfusion. In the present treatment of liver injury, synthetic drugs such as corticosteroids and IFN are commonly employed to manage the progression of this condition (108). When liver injury is exacerbated and progresses to liver failure, liver transplantation emerges as one of the limited therapeutic alternatives. This approach faces numerous challenges, with the low graft success rate being a prominent issue (109). Natural compounds with anti-inflammatory and antioxidant properties offer significant protection against liver injury (110,111). Among them, AS-IV, a typical natural compound, possesses strong anti-inflammatory and antioxidant activity and shows great potential for use in liver injury prevention (112).

As a discipline focusing on drug absorption, distribution and metabolism excreted in living organisms, pharmacokinetics is crucial for understanding drug action and efficacy. AS-IV presents a unique pharmacokinetic profile because of its own characteristics, such as low intestinal permeability, a relatively high molecular weight, and limited lipophilicity, which is associated with paracellular transport.

The oral bioavailability of AS-IV remains low across different species. When orally given to rats and beagles at 10 mg/kg dose, its in vivo bioavailability was only 3.66 and 7.4%, respectively. The low oral bioavailability substantially limits systemic drug exposure, making it challenging to reach therapeutically effective concentrations in target tissues. Therefore, intravenous injection is generally adopted for AS-IV administration in clinical settings. However, when the drug concentration in various tissues was measured subsequent to the intravenous injection of AS-IV to rats, the liver and kidneys exhibited the highest levels of AS-IV, followed by the lungs, heart and spleen. By contrast, the limited distribution of AS-IV to the brain indicates that it might encounter challenges in crossing the blood-brain barrier. Within the concentration range of 250-1,000 ng/ml, ~90% of AS-IV was bound to plasma proteins. In plasma, AS-IV primarily exists in a bound state, with relatively low free drug concentrations (149,150).

The elimination of AS-IV exhibits certain variations across different species yet displays overall linear kinetic characteristics. For instance, following the intravenous injection of 0.5, 1 and 2 mg/kg of AS-IV in beagles, the plasma t_1/2 was 177.18, 196.58 and 241.59 min, respectively, and the area under the curve (AUC) was 126.24, 276.28 and 724.51 µg h/ml respectively (149). By contrast, when AS-IV was administered intravenously only to healthy volunteers, the mean maximum plasma concentration (C_max) values were 2.12, 3.59, 3.71 and 5.17 g/ml at single doses of 200, 300, 400 and 500 ml (contained 27, 36, 45 and 54 mg AS-IV, respectively), respectively. The corresponding mean values of AUC were 4.38, 9.75, 13.59 and 18.22 µg h/ml, respectively, and the mean values of elimination t_1/2 were 2.14, 2.59, 2.62 and 2.69 h, respectively (151).

In terms of excretion, AS-IV is primarily eliminated via feces and bile, with urinary excretion accounting for a relatively minor proportion. For male rats, the excretion rates of the substance in feces, urine and bile within 24 h were 31.41, 13.43 and 31.92%, respectively. For female rats, the corresponding excretion rates in feces, urine and bile over the same 24-h period were 31.84, 21.77 and 36.20%, respectively (150). In healthy volunteers, the cumulative urinary excretion of AS-IV over 24 h after administration of 500 ml was 3.91% (151). However, in rat models, only 50% of AS-IV was metabolized in vivo via urine and feces, 30% was recovered in bile, and metabolism was virtually non-existent in the liver, suggesting that first-pass elimination does not occur (47,152).

Understanding the toxic impacts of AS-IV is essential for the development of AS-IV-related drugs. For therapeutic applications, AS-IV has a high safety profile, with low levels of toxicity at standard doses that do not induce significant adverse effects. Oral administration of AS-IV at a daily dose of 1.0 mg/kg for 14 weeks did not cause adverse effects on hepatic and renal function (153). Animal studies also confirmed that AS-IV treatment does not lead to toxicity or side effects in adult animals. The safe doses for rats and beagles are equivalent to 70 or 35-fold the human dose, respectively, such as 0.57 g/kg (154). When AS-IV was administered intravenously at a daily dose of 1.0 mg/kg, it caused maternal toxicity in rats, and doses >0.5 mg/kg led to fetal toxicity. By contrast, when AS-IV was administered intravenously daily from 4 weeks before mating until day 6 of pregnancy in rats, no maternal toxicity was observed at doses ranging from 0.25-1.0 mg/kg. However, at the highest dose of 1.0 mg/kg, suppression of female fertility and delayed hair growth and neurological development in the offspring were observed. These findings indicate that while the toxic effects of AS-IV vary markedly with the stage of pregnancy, the compound consistently lacks teratogenic effects, underscoring the need for caution in its clinical use during pregnancy (155,156). Therefore, caution should be exercised when applying AS-IV treatment in the pregnant population.

However, current research on the adverse reactions and toxic side effects of AS-IV remains limited. Most findings are based on preliminary observations, and the underlying mechanisms of its potential toxicity have not been fully elucidated. Notably, long-term human clinical data are still lacking, rendering it difficult to confirm its long-term safety profile in chronic conditions such as viral liver disease. Therefore, clinical studies on the toxicity of AS-IV in liver disease are urgently needed to facilitate its development and application in the therapeutic field of liver disease.

AS-IV suffers from poor permeability and low oral bioavailability. Nanocapsules and nanocarriers have been effective in enhancing drug bioavailability (157). In terms of brain-targeted delivery, the use of the ion cross-linking method for β-asarone-modified asi-loaded chitosan nanoparticles with 120 nm size and favorable uniformity led to ~20% cumulative release in vitro over 48 h, demonstrating sustained-release properties. Compared with unmodified nanoparticles, DiR-βCS-NP exhibited 1.52-fold enhanced uptake in 16HBE cells and 2.49-fold increased fluorescence intensity in the brain, significantly improving nasal brain targeting efficiency. ASI-βCS-NP reduced clinical scores in EAE mice by ~60%, inhibited glial cell activation, mitigated myelin loss and promoted myelin regeneration, demonstrating superior nasal-brain delivery and neuroprotective effects (158). In terms of stability and hydrophilicity, encapsulation in liposomes and loading them onto PVA nanofibers yielded particles with an average diameter of 143.23±3.25 nm and a polydispersity index of 0.11±0.048. The zeta potential was -11.2±1.35 mV, indicating uniform particle distribution without significant aggregation. The ASL/APS/PVA nanofiber surface exhibited smoothness and structural stability, protecting AS-IV from degradation while enhancing its storage and in vivo transport stability. Furthermore, the liposomes encapsulated the hydrophobic AS-IV, enabling its stable dispersion in the aqueous phase. The PVA nanofibers demonstrated excellent hydrophilicity, adsorbing bodily fluids to promote AS-IV dispersion and sustained release within aqueous environments, and significantly improving its hydrophilic properties (159). In tumor-targeted drug delivery, nanomicelles efficiently encapsulate AS-IV with a 90.92% encapsulation efficiency and 12.48% loading capacity, significantly enhancing its water solubility and dispersibility. This nanomedicine exhibited stable particle size in serum and at 4°C storage conditions, demonstrating favorable biosafety. Following PDL1 modification, the tumor-targeting capability was enhanced, enabling the precise delivery of AS-IV and inhibition of the STAT3/NF-κB pathway, substantially boosting antitumor efficacy (160). In addition, AS-IV can enhance ocular delivery. AS-IV-loaded lipid nanocapsules (LNCs) with uniform particle sizes of 19.88, 49.39 and 92.89 nm showed an encapsulation efficiency of over 90%. AS-IV-LNCs-20 also markedly reduced retinal apoptosis rate from 5.12 to 0.533% and alleviated ROS accumulation, while showing no ocular irritation and excellent biocompatibility (161). Nanoparticles, nanomicelles and LNCs are excellent in tissue-targeted delivery; and the liposome-PVA nanofiber system has advantages in improving stability and water solubility. These results demonstrate that nanotechnology greatly enhances the solubility, stability, ocular penetration and retinal targeting efficiency of AS-IV, providing new ideas and ways to improve the pharmacokinetic properties of AS-IV.