The Dual Role of Saikosaponins in Liver Disease Treatment: A Comprehensive Review of Pharmacological Activities and Toxicological Characteristics

Abstract

Liver diseases, including viral hepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma (HCC), remain major global health burdens due to their high prevalence and limited therapeutic options. The need for safer and more effective hepatoprotective agents has renewed interest in traditional herbal medicines such as Radix Bupleuri. Its major bioactive constituents, saikosaponins (SSs), exhibit diverse pharmacological activities. This review synthesizes recent advances in the pharmacodynamics, molecular mechanisms, and toxicological characteristics of SSs, emphasizing their dual hepatoprotective and hepatotoxic properties. Relevant literature published from 2000 to 2025 was systematically retrieved from major scientific databases, including PubMed, Web of Science, Google Scholar, and other sources as appropriate, with emphasis on mechanistic studies and in vitro/in vivo evidence. SSs exert hepatoprotective effects through multiple mechanisms, including inhibition of nuclear factor κB (NF-κB) and signal transducer and activator of transcription 3 (STAT3) signaling pathways, activation of nuclear receptors, induction of hepatic stellate cell apoptosis and autophagy, and modulation of lipid metabolism via peroxisome proliferator-activated receptor α (PPARα)/sterol regulatory element-binding protein 1c (SREBP1c) signaling. SSs in HCC inhibit Cyclooxygenase-2 and STAT3, promote apoptosis and ferroptosis, suppress angiogenesis, and enhance chemotherapy and radiotherapy sensitivity. However, accumulating evidence indicates that SSs may induce dose-dependent hepatotoxicity through oxidative stress, apoptosis and autophagy injury. These dual pharmacological effects are influenced by CYP regulation, bioavailability, and potential drug-drug interactions. Overall, SSs represent promising yet complex therapeutic candidates. Optimization of dosing strategies, clarification of mechanistic determinants, and development of advanced delivery systems are essential for their safe clinical translation. Future research should incorporate multi-omics approaches, physiologically relevant liver models, and rigorously designed clinical trials to establish standardized Saikosaponin-based therapies for liver diseases.

1. INTRODUCTION

Liver diseases encompass a broad spectrum of pathological conditions, including viral hepatitis, alcoholic liver disease, metabolic-dysfunction-associated fatty liver disease (MAFLD), liver fibrosis, cirrhosis, and hepatocellular carcinoma (HCC).^1,2) These conditions represent significant global health challenges due to their high morbidity and mortality rates.^1,2) Chronic hepatitis, often caused by hepatitis B and C viruses, leads to progressive liver damage and fibrosis. Cirrhosis, the end-stage of chronic liver disease, is characterized by extensive fibrosis and architectural distortion of the liver parenchyma, resulting in impaired liver function and portal hypertension.^3–5) HCC is a primary malignancy arising predominantly in cirrhotic livers and remains a leading cause of cancer-related deaths worldwide.^2,3)

Current therapeutic strategies vary according to disease etiology and stage. Antiviral therapies have significantly improved outcomes in viral hepatitis. For cirrhosis and its complications, management includes pharmacological interventions such as terlipressin for variceal bleeding and hepatorenal syndrome, as supported by recent clinical practice guidelines.^2,6) Statins have emerged as potential agents to reduce portal hypertension and fibrogenesis in chronic liver disease, although their long-term effects require further investigation.^4,7) For HCC, treatment options include surgical resection, locoregional therapies, and systemic chemotherapy, with ongoing research to improve survival outcomes.⁸⁾ Despite advances, challenges remain in early diagnosis, effective treatment, and management of adverse effects, underscoring the need for novel therapeutic agents with hepatoprotective efficacy and safety.

Objectives saikosaponins (SSs) constitute a class of medicinal monomers characterised by a triterpene tricyclic structure.⁹⁾ Modern high-efficiency separation techniques such as ultra-high performance supercritical fluid chromatography have been employed for the separation and quantification of SSs. The primary subclasses of SSs include saikosaponin a (SSa), saikosaponin b1, saikosaponin b2 (SSb2), saikosaponin c (SSc), saikosaponin d (SSd), among others^9–12) (Fig. 1). SSs, particularly SSa and SSd, have demonstrated significant hepatoprotective effects in various experimental models of liver injury and disease. These effects primarily include anti-inflammatory, antioxidant, antifibrotic, and antitumor activities, rendering them highly promising candidate drugs in the field of liver disease therapy.^12,13)

Fig. 1. Chemical Structures of Key Saikosaponin Subclasses

SSa has been shown to ameliorate liver injury induced by lipopolysaccharide (LPS) and D-galactosamine (D-GalN) in mice by activating liver X receptor alpha (LXRα), which leads to suppression of the nuclear factor κB (NF-κB) signaling pathway and reduction of pro-inflammatory cytokines such as tumor necrosis factor α (TNF-α) and interleukin-1β (IL-1β). This results in decreased oxidative stress and inflammatory damage in hepatic tissue.¹⁴⁾ Our research has also demonstrated that pretreatment with SSd can attenuate the inflammatory response in a rat model of hepatic ischemia–reperfusion injury, and this effect may be associated with the downregulation of toll-like receptor 4/NF-κB signaling pathway proteins.¹⁵⁾ These findings suggest that SSa and SSd exerts protective effects by modulating nuclear receptor pathways and inflammatory responses. SSd exhibits anti-fibrotic properties by inducing apoptosis in hepatic stellate cells (HSCs), the key effector cells in liver fibrosis. SSd triggers caspase–3–dependent and independent cell death pathways and induces mitochondrial injury through BAX/BAK activation, ultimately reducing activated HSC levels and thereby mitigating hepatic fibrogenesis.¹⁶⁾ Additionally, SSd alleviates liver fibrosis by negatively regulating reactive oxygen species (ROS) and the NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome via activation of the estrogen receptor beta (ERβ) pathway, further contributing to its anti-inflammatory and anti-fibrotic effects.¹⁷⁾

Recent studies also highlight the lipid-lowering effects of SSs in hyperlipidemic models, where they enhance hepatic lipid and cholesterol metabolism, promote fatty acid oxidation, and reduce oxidative stress and inflammation associated with lipid overload.¹⁸⁾ These metabolic regulatory effects may indirectly benefit liver health by mitigating steatosis and related liver injury. Clinically, formulations containing SSs, such as Sho-saiko-to, have been used in traditional medicine for chronic liver diseases. Clinical trials have demonstrated that Sho-saiko-to can reduce hepatocyte necrosis, inhibit hepatic fibrosis, and lower the incidence of HCC in cirrhotic patients.¹⁹⁾ However, adverse effects including interstitial pneumonia have been reported, particularly with long-term use or in combination with interferon, indicating the need for careful monitoring.^19,20)

In summary, these findings highlight the multifaceted hepatoprotective potential of SSs and their subclasses, while underscoring the need for enhanced monitoring of hepatotoxicity, thereby providing a supportive basis for further research and development in clinical applications for liver disease treatment.

2. HEPATOPROTECTIVE EFFECTS OF SAIKOSAPONINS AND THEIR SUBCLASSES

2.1. Anti-hepatitis Effects and Mechanisms

When damage-associated molecular patterns are released following hepatocyte injury, sterile inflammation occurs in the liver. This activates pattern recognition receptors in monocytes, Kupffer cells, and neutrophils, leading to the massive production and release of pro-inflammatory cytokines such as IL-1β, interleukin-18 (IL-18), and TNF-α. Ultimately, these processes result in hepatic inflammation and cell death.²¹⁾

SSs have demonstrated significant anti-hepatitis activities across various experimental models. Liu et al.²²⁾ induced acute liver injury in mice with acetaminophen (APAP), increasing hepatocellular injury markers alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and inflammatory markers signal transducer and activator of transcription 3 (STAT3), p-STAT3, and NF-κB phospho-p65 (p-p65). SSd pretreatment reduced these indicators and upregulated IL-10, showing that SSd alleviates injury by inhibiting NF-κB and STAT3, with similar effects in vitro. In CCl₄-induced HL-7702 cells, SSd downregulated NLRP3, caspase-1, IL-1β, IL-18, and HMGB1, indicating modulation of inflammatory responses via the NLRP3 inflammasome.²³⁾ Regarding SSa, current experiments confirm that its anti-inflammatory effects are primarily associated with the inhibition of NF-κB signaling pathway activation, upregulation of LXRα expression, and activation of the LXRα-dependent cholesterol efflux pathway, thereby achieving hepatoprotective effects.^14,24)

In addition, SSs exert multi-pathway anti-inflammatory effects by modulating nicotinic acid and nicotinamide metabolism (possibly through increased oxidized form of nicotinamide adenine dinucleotide synthesis and sirtuin 1 activation to suppress the NF-κB pathway) and arachidonic acid metabolism (inhibiting cyclooxygenase (COX)/lipoxygenase pathways to reduce the production of inflammatory mediators such as prostaglandins and leukotrienes). Their efficacy is comparable to aspirin with fewer side effects, demonstrating potential clinical application value.²⁵⁾ Notably, studies have shown that SSb2 inhibits hepatitis C virus (HCV) through multi-target mechanisms and can overcome daclatasvir-induced nonstructural 5A resistance mutations, offering new insights for subsequent hepatitis virus suppression.²⁶⁾

2.2. Anti-hepatic Fibrosis and Cirrhosis Effects and Mechanisms

Alpha-smooth muscle actin (α-SMA) acts as a diagnostic indicator for the activation of HSCs, while transforming growth factor β1 (TGF-β1) is a pro-fibrogenic agent secreted by these activated HSCs.²⁷⁾ The activation of HSCs is crucial in the development of fibrosis, leading to the excessive accumulation of extracellular matrix (ECM).²⁸⁾ Key factors driving HSC activation include hepatocyte injury and the activation of Kupffer cells.²⁹⁾ Research indicates that damage to hepatocytes, along with the phosphorylation of NF-κB and p38 mitogen-activated protein kinase (p38 MAPK), can instigate HSC activation and the abnormal secretion of ECM, thereby facilitating hepatic fibrosis.³⁰⁾ Conversely, activated Kupffer cells produce significant amounts of ROS, which enhance the expression of TGF-β1 and, in turn, induce the activation of HSCs.^31,32)

The anti-fibrotic effects of SSs in hepatic fibrosis and cirrhosis models have been extensively studied. SSs (particularly SSd) can effectively alleviate hepatic fibrosis and cirrhosis by modulating hepatic stellate cell activation, promoting autophagy and apoptosis, and regulating key signaling pathways in lipid metabolism and inflammation.

SSd induces apoptosis and autophagy in activated HSCs, thereby reducing ECM deposition and fibrosis progression.³³⁾ Molecular studies have elucidated that SSd modulates the ERβ pathway to exert its anti-fibrotic effects. Activation of ERβ by SSd suppresses oxidative stress-induced HSC activation and downregulates profibrotic markers such as TGF-β1, collagen type I, and tissue inhibitor of metalloproteinases-1 (TIMP-1), while upregulating matrix metalloproteinase-1 (MMP-1), which promotes ECM degradation.³⁴⁾ Furthermore, SSd negatively regulates the ROS/NLRP3 inflammasome pathway via ERβ activation, reducing inflammation and fibrogenesis in the liver.¹⁷⁾

In vivo studies using CCl₄-induced liver fibrosis models have confirmed that SSd treatment significantly reduces liver fibrosis and inflammation. SSd also modulates the G protein-coupled estrogen receptor 1 (GPER1)/autophagy signaling pathway, contributing to its anti-fibrotic effects.³⁵⁾ Liposome-encapsulated SSd formulations have been developed to enhance therapeutic efficacy and reduce toxicity, showing improved survival rates and liver tissue repair in fibrotic mice compared to pure SSd.³⁶⁾

Experiments conducted by Chen et al.³⁷⁾ indicated that SSd significantly diminishes the expression levels of platelet-derived growth factor (PDGF), TGF-β1, the TGF-β1 receptor, and α-SMA in HSC-T6 cells, while also promoting apoptosis in these cells, thereby effectively curbing liver fibrosis. Subsequent investigations have elucidated that the apoptosis induction mechanism employed by SSd in HSC-T6 cells may involve pathways that are caspase-3-dependent, caspase-3-independent, as well as mitochondrial in nature.¹⁶⁾

Recently, SSd has been reported to induce autophagosome formation, which can inhibit HSC activation and proliferation by upregulating GPER1 and microtubule-associated protein light chain 3 II (LC3-II) expression.^35,38) These studies elucidate the upstream molecular mechanisms by which SS inhibits HSC proliferation and promotes HSC death.

Other SSs, such as SSa, have demonstrated hepatoprotective effects in models of liver injury associated with fibrosis by activating LXRα, which regulates lipid metabolism and inflammation.¹⁴⁾ Furthermore, bone morphogenetic protein 4 (BMP-4) is a crucial growth factor implicated in liver fibrosis and carcinogenesis. Experimental studies have confirmed that SSa downregulates BMP-4 expression and induces apoptosis in HSCs, suggesting the potential therapeutic application of SSa for liver diseases characterized by elevated BMP-4 expression.^37,39,40)

However, although the effects of SSs on activated HSCs contribute substantially to their anti-fibrotic efficacy, a recent study by Sugimoto et al.⁴¹⁾ revealed that HSCs also exert essential physiological functions in non-fibrotic livers, including the regulation of hepatocyte zonation, metabolic homeostasis, and regeneration, as evidenced by findings that HSC deficiency disrupts liver zonation and exacerbates alcohol-associated and MAFLD. Therefore, long-term or non-selective SS-induced HSC apoptosis in non-fibrotic livers may theoretically impair hepatic homeostasis. Current data suggest that SSs-mediated apoptosis primarily occurs in activated HSCs; however, systematic evaluation of their long-term effects in non-fibrotic settings remains limited. Future studies should clarify activation-state specificity and optimize targeted strategies to balance anti-fibrotic efficacy with hepatic safety.

2.3. Anti-hepatocellular Carcinoma Effects and Mechanisms

SSs exhibit promising anti-HCC activities through diverse mechanisms. The etiology of HCC includes viral hepatitis, liver cirrhosis, MAFLD, alcoholic hepatitis, tumor suppressor gene TP53 mutations, among others, while aspirin has been demonstrated to have preventive effects against HCC.⁴²⁾ COX-2 overexpression has been widely reported in HCC and is considered an early event during hepatocarcinogenesis.⁴³⁾ In a diethylnitrosamine (DEN)-induced rat liver cancer model, Lu et al.⁴⁴⁾ demonstrated that SSd reduces the high expression of COX-2 in both tumor cells and macrophages of liver tissues, confirming that SSd prevents hepatocarcinogenesis by inhibiting COX-2. This indicates that SSd exhibits pharmacological activity similar to that of nonsteroidal anti-inflammatory drugs, thereby providing experimental evidence for its clinical application in cancer therapy. Ren et al.⁴⁵⁾ further demonstrated that SSd inhibits STAT3 phosphorylation and downregulates CCAAT/enhancer binding protein beta (C/EBPβ) expression, leading to reduced COX-2 expression. They proposed that SSd suppresses hepatocarcinogenesis via the p-STAT3/C/EBPβ/COX-2 signaling pathway. Additionally, SSd can downregulate COX-2 expression by inhibiting the p-STAT3/ hypoxia-inducible factor-1α (HIF-1α) signaling pathway.⁴⁶⁾

SSd has been shown to inhibit proliferation and induce apoptosis in HCC cell lines such as HepG2 and Hep3B. The pro-apoptotic effects involve upregulation of p53 and p21/WAF1, activation of Fas/Fas ligand pathways, and modulation of B-cell lymphoma 2 (Bcl-2) family proteins, leading to cell cycle arrest at the G1 phase and enhanced apoptotic cell death.⁴⁷⁾ Other SSs, such as SSa and SSb2, have demonstrated anti-angiogenic effects by downregulating vascular endothelial growth factor (VEGF)/extracellular signal-regulated kinase (ERK)/HIF-1α signaling, thereby inhibiting tumor angiogenesis essential for HCC progression.⁴⁸⁾ SSs also induces ferroptosis, a form of regulated cell death characterized by iron-dependent lipid peroxidation, through endoplasmic reticulum stress-mediated activation of ATF3 and suppression of SLC7A11 expression in HCC cells.⁴⁹⁾

SSs have been studied for its potential to enhance the efficacy of radiotherapy and chemotherapy in treating liver diseases, particularly HCC, and to modulate drug resistance mechanisms. Studies have demonstrated that SSd enhances the radiosensitivity of HCC cells by inducing autophagy, thereby increasing cell mortality following radiotherapy.⁵⁰⁾ Wang et al.⁵¹⁾ posited that SSd enhances the radiosensitivity of SMMC7721 HCC cells and triggers apoptosis, likely through the activation of the p53/Bcl2 signaling pathway and the modulation of G0/G1 and G2/M cell cycle checkpoints. Subsequent investigations by Wang’s team revealed that under hypoxic conditions, SSd downregulates HIF-1α expression while upregulating p53 and BAX expression, concurrently decreasing Bcl-2 expression, indicating SSd’s potential role as a sensitizer in the radiotherapy of HCC.⁵²⁾ Furthermore, SSd fosters autophagy formation and curtails the growth of HCC cells by inhibiting mammalian target of rapamycin (mTOR) phosphorylation, thereby augmenting radiosensitivity and hindering proliferation. These inhibitory effects can be partially reversed by the autophagy inhibitor chloroquine or an mTOR agonist.⁵³⁾ Recent research suggests that SSd elevates the expression of small ubiquitin-like modifier-specific protease 5 (SENP5), suppresses small ubiquitin-like modifier 1 and Gli1 expression, enhances chemosensitivity, and impedes the proliferation of HCC cells.⁵⁴⁾

Studies indicate that SSs can act as chemosensitizers, improving the effectiveness and selectivity of anticancer drugs. For instance, SSa enhances gemcitabine sensitivity in intrahepatic cholangiocarcinoma by modulating the phospho-protein kinase B (p-AKT)/BCL-6/ATP-binding cassette transporter A1 (ABCA1) signaling pathway, leading to increased apoptosis and reduced tumor progression.⁵⁵⁾ This combination strategy may overcome chemoresistance and improve therapeutic outcomes. SSb2 influences the activity and expression of multidrug resistance-associated transporters such as P-glycoprotein (Pgp), multidrug resistance-associated proteins (MRP1, MRP2), and organic cation transporter 2. By inhibiting these transporters in an environment-dependent manner, SSb2 enhances the hepatotargeting and intracellular accumulation of anticancer drugs, potentially increasing their efficacy while reducing systemic toxicity.⁵⁶⁾ These findings suggest that SSs may serve as valuable adjuvants in combination therapies for liver cancer.

However, the potential for drug interactions necessitates careful evaluation. SSs can influence CYP enzymes and drug transporters, which may alter the pharmacokinetics of co-administered drugs.⁵⁷⁾ Therefore, further studies are required to elucidate the safety and optimal use of SSs in combination regimens.

In summary, SSs exert anti-HCC effects through multiple pathways, including inhibiting COX-2 expression, inducing apoptosis and ferroptosis, blocking angiogenesis, and enhancing sensitivity to radiotherapy and chemotherapy, supporting their potential as adjuvant therapeutic agents for liver cancer.

2.4. Hepatoprotective Effects in Other Liver Diseases

Beyond hepatitis, fibrosis, and cancer, SSs have demonstrated protective effects in various other liver diseases, including drug-induced liver injury (DILI) and MAFLD.

Oxidative stress refers to the disruption of the balance between the generation of ROS and antioxidant defenses, leading to tissue and cellular damage.⁵⁸⁾ When oxidative stress occurs, indicators reflecting oxidative stress levels—such as glutathione peroxidase, superoxide dismutase (SOD), and catalase—decrease, while the lipid peroxidation marker malondialdehyde (MDA) increases.⁵⁹⁾ In models of drug-induced liver injury, SSs exhibit antioxidant and anti-inflammatory properties. For example, SSd reduces cyclophosphamide-induced hepatotoxicity by modulating cytokine networks, apoptotic pathways, and oxidative stress markers, thereby attenuating liver damage.⁶⁰⁾ Lin et al.²³⁾ induced acute injury in HL-7702 cells using CCl₄, manifested by increased levels of ALT, AST, and MDA, and decreased SOD activity, while SSd reversed these effects. This indicates that SSd can inhibit CCl₄-induced oxidative stress damage in hepatocytes. SSd also exhibited antioxidative stress effects in a CCl₄-induced acute liver injury model in mice.⁶¹⁾

MAFLD, formerly known as non-alcoholic fatty liver disease, is a chronic liver condition characterized by hepatic steatosis in ≥5% of hepatocytes, excluding other causes such as excessive alcohol consumption.⁶²⁾ MAFLD encompasses a spectrum of liver disorders ranging from simple steatosis to steatohepatitis, which may progress to cirrhosis and HCC.⁶³⁾

SSd ameliorates MAFLD by modulating lipid metabolism through coordinated regulation of peroxisome proliferator-activated receptor alpha (PPARα) activation and inhibition of sterol regulatory element-binding protein 1c (SREBP1c)-mediated fatty acid synthesis. This dual action promotes fatty acid oxidation and reduces lipid accumulation in hepatocytes.⁶⁴⁾ SSd also reduces oxidative stress and inflammation associated with fatty liver disease, contributing to improved liver function.

As summarized in Table 1, SSs particularly SSd and SSa, exert broad hepatoprotective effects in hepatitis, fibrosis, HCC, DILI, and MAFLD models through coordinated regulation of inflammation (NF-κB/STAT3/NLRP3), fibrogenesis (TGF-β1/α-SMA), tumor-related pathways (COX-2/HIF-1α/mTOR/p53), oxidative stress (ROS), and lipid metabolism (PPARα/SREBP1c), highlighting their multi-target therapeutic potential in liver diseases.

Table 1. Hepatoprotective Effects and Molecular Mechanisms of Saikosaponins

Pharmacological effects	SSs	Experimental model	Dosage	Key findings	Ref.
Anti-Hepatitis Effects	SSd	C57BL6 mice (APAP-induced liver injury)	2 mg/kg/d (i.p.)	NF-kB, STAT3, IL-6 and Ccl2↓, IL-10↑	22)
	SSd	HL7702 cells	0.5–2 µM	NLRP3, Caspase-1, IL-1β, IL-18 and HMGB1↓	23)
	SSa	C57BL/6 mice (LPS/D-GalN-induced liver injury)	5–20 mg/kg (i.p.)	LXRα↑. MPO, MDA, AST, ALT, NF-κB, TNF-α and IL-1β↓	14)
	SSb2	Huh-7.5.1 and 293T cells	10 and 100 µM	inhibits HCV entry, polyprotein translation, and RNA replication	26)
	SSb2	HuH7 cells and their derivatives HuH7.5 and S29 cells	50 µM	Neutralization of virus particles, viral attachment, viral entry/fusion, binding of serum-derived HCV onto hepatoma cells	65)
	SSc	2.2.15 cell line	2.5–40 µg/mL	HBsAg, HBeAg and HBV DNA↓	66)
Anti-Hepatic Fibrosis	SSd	LX-2 and HSC-T6 cells	1 µM	caspase-3 and caspase-9↑	16)
	SSd	C57BL/6 mice (CCl4-induced liver fibrosis)	2 mg/kg (i.p.)	ERβ↑. ROS, NLRP3, IL-1, IL-18, α-SMA and TGF-β1↓	17)
	SSd	LX-2 cells	5 µM	α-SMA, NLRP, pro-IL-β1, IL-1b, and IL-18↓	17)
	SSd	C57 mice (CCl4-induced liver fibrosis)	2 mg/kg (i.p.)	α-SMA, Col 1 and BECN1↓. p62↑	35)
	SSd	LX-2 cells	5 µM	LC3-II↓. p62, ERβ and GPER1↑	35)
	SSd	HSC-T6 cells	10 µmol/L	cleaved caspase 3, BAX and LC3-II↑. Ki67 and Bcl2↓	33)
	SSd	HSC-T6 cells	5 µM	TGF-β1, Hyp, COL1 and TIMP-1↓. MMP-1↑	34)
	SSd	C57BL/6 mice (The thioacetamide -induced mouse model of hepatic fibrosis)	20–50 mg/kg (i.p.)　 1–4 µM	Lipo-SSd is more effective than pure SSd in ameliorating liver fibrosis	36)
	SSd	HSC-T6 cells	1 µM	PDGFR1, TGF-b1R, a-SMA, CTGF and TGF- β1↓	37)
	SSd	HSC-T6 and LX-2 cells	2.5 µM	α-SMA and BMP-4↓	40)
	SSa	HSC-T6 and LX-2 cells	5 µM	α-SMA and BMP-4↓. BAX↑	40)
	SSa	HSC-T6 cells	10 µM	PDGFR1, TGF-β1R, α-SMA, CTGF and TGF-β1↓	37)
	SSa	HSC-T6 and LX-2 cells	10 µM	Apaf-1, CytC, EndoG and AIF↑	39)
	SSa	SD rat (CCl4-induced liver fibrosis)	0.004% (i.g.)	TNF-α, IL-1β, IL-6, TGF-β1, NF-κB and Hydroxyproline↓. IL-10↑	67)
Anti-HCC Effects	SSd	SD rat (DEN-induced HCC model)	2 mg/kg (i.p.)	C/EBPβ and COX-2↓.	44)
	SSd	SMMC-7721 and HepG2 cells	2.5–15 µg/mL	COX-2↓. p-STAT3/C/EBPβ↓	45)
	SSd	SMMC-7721 and HepG2 cells	2.5–15 µg/mL	COX-2↓. p-STAT3/HIF-1α↓	46)
	SSd	HepG2 and Hep3B cells	2.5–5 µM	p53↑, p21/WAF1↑, Fas/APO-1↑, Bax↑, IκBα↑.	47)
	SSd	SMMC-7721 and MHCC97L cells	3 µg/mL	mTOR↓. LC3-II↑. Radiosensitization	50)
	SSd	SMMC-7721 cells	1 and 3 µg/mL	Bcl2↓. P53 and BAX↑. Regulating the G0/G1 and G2/M checkpoints enhances the radiosensitivity of HCC cells	51)
	SSd	SMMC-7721 and HepG2 BALB/c nude mice (SMMC-7721 cell xenograft tumor model)	1–20 µg/mL　 0.75 mg/kg (i.p.)	HIF-1α and Bcl2↓. P53and BAX↑. Radiosensitization	52)
	SSd	SMMC-7721 and MHCC97L	3 µg/mL	mTOR↓. p62↓. LC3-II and Beclin-1↑,	53)
	SSd	Hep3B cells	2–15 µM	SENP5↑. inhibited Gli1 SUMOylation, suppressed the malignant phenotype of HCC cells, and enhanced chemosensitivity	54)
	SSd	Hep G2 cells	2.5–10 µg/mL	caspases 3 and 7↑	66)
	SSd	SD rats (N-diethylnitrosamine-induced HCC model)	1.5 mg/kg (i.p.)	syndecan-2, MMP-2, MMP-13 andTIMP-2↓	68)
	SSd	HepG2 cells	10 µM	NF-κB↓	69)
	SSd	SMMC-7721	3 µg/mL	MDA↓, GSH↑, radiosensitizer	70)
	SSa	the human cholangiocarcinoma cell lines HCCC-9810 and RBE. Mouse (Patient-Derived Xenografts)	5 µM　 10 mg/kg (i.p.)	Enhancing the sensitivity of cholangiocarcinoma cells to gemcitabine through the p-AKT/BCL6/ABCA1 signaling axis	55)
	SSa	HepG2 and Huh-7 cells	20 µM	SLC7A11 and GSH↓. ATF3 and MDA↑	49)
	SSa	BALB/c mice (Subcutaneous injection of Huh-7 cells)	1 and 3 mg/kg (i.g)	ATF3↑. SLC7A11↓	49)
	SSa	HepG2 cells	10 and 12.5 µg/mL	p-15INK4b↑, p-16INK4a↑, Sensitizing drug-resistant cancer cells	71)
	SSa	HepG2/ADM cells	5 µM	Apoptosis, retention, chemosensitivity of P-gp overexpressing HepG2/ADM cells to DOX, VCR and paclitaxel↑	72)
	SSb2	BALB/c mice (through inoculation with H22 hepatoma cells). HepG2	5–20 mg/kg/d (i.p.)　 15–60 µg/mL	VEGF/ERK/HIF-1α↓	48)
	SSb2	BRL3A cells	64 and 128 µM	Environment-dependent inhibition of multidrug resistance-associated drug transporters (Pgp, MRP1, and MRP2)	56)
Other hepatoprotective effects	SSd	ICR mice (CCl4-induced acute liver injury )	1, 1.5, and 2 mg/kg (i.p.)	oxidative stress and NLRP3↓	61)
	SSd	C57BL/6J mice. HepG2 cells	5, 10 and 20 mg/kg (i.g)　 0–20 µM	PPARα, ACOX1, CPT1α and INSIG1/2↑, SREBP1c↓	64)

α-SMA Alpha-smooth muscle actin, Acox1 acyl-CoA oxidase 1, AIF apoptosis inducing factor, APAP acetaminophen, Apaf-1 apoptotic protease activating factor-1, BAX Bcl-2-associated X protein, BMP-4 bone morphogenetic protein 4, C/EBPβ CCAAT/enhancer binding protein beta, COX-2 Cyclooxygenase-2, Cpt1α carnitine palmitoyltransferase 1α, CTGF connective tissue growth factor, CytC cytochrome c, EndoG endonuclease G, ER endoplasmic reticulum, ERβ estrogen receptor beta, GPER1 G protein-coupled estrogen receptor 1, GSH glutathione, HCC hepatocellular carcinoma, HIF-1α hypoxia-inducible factor-1α, HSCs hepatic stellate cells, i.g. intragastric administration, i.p. intraperitoneal injection, INSIG insulin induced gene, LC3-II light chain 3 II, LPS/D-GalN lipopolysaccharide and D-galactosamine, LXRα liver X receptors α, MAFLD metabolic-dysfunction-associated fatty liver disease, MDA malondialdehyde, MMP matrix metalloproteinase, PDGF platelet-derived growth factor, Pgp P-glycoprotein, PPARα peroxisome proliferator-activated receptor alpha, ROS reactive oxygen species, SENP5 SUMO specific peptidase 5, SREBP1c sterol regulatory element-binding protein 1c, SS Saikosaponin, SSs Saikosaponins, TGF-β1 transforming growth factor β1, TIMP tissue inhibitor of metalloproteinases, TOR mechanistic target of rapamycin, VEGF vascular endothelial growth factor.

3. RESEARCH ON THE HEPATOTOXICITY OF SAIKOSAPONINS AND THEIR SUBCLASSES

3.1. Induction of Oxidative Stress Injury

The administration of SSs (particularly SSd) induces dose- and time-dependent liver injury, characterized by elevated serum aminotransferases (AST, ALT) and lactate dehydrogenase, indicating hepatocellular damage. Li et al.⁷³⁾ found that saikosaponin-treated mice exhibited an increased liver-to-body weight ratio and presented histopathological alterations such as hepatocyte apoptosis and inflammatory infiltration. Proteomic analysis revealed that SSs administration upregulated the expression of MDA, ROS, and CYP2E1 (an enzyme associated with oxidative stress generation), while downregulating GSH expression levels, thereby exacerbating liver injury through ROS production and lipid peroxidation. Experimental results from Li et al.⁷⁴⁾ further confirmed that SS suppresses CYP3A4 levels, reduces SOD activity, and increases MDA levels, suggesting that its hepatotoxicity is associated with the hepatic CYP enzyme system and oxidative damage mechanisms.

3.2. Induction of Apoptosis Injury

SSd has been demonstrated to activate the extrinsic apoptosis pathway in human hepatocytes. Chen et al.⁷⁵⁾ illustrated that SSd diminishes the viability of HL-7702 cells at a concentration of 0.4 µmol/L. The induction of apoptosis in HL-7702 hepatocytes by SSd occurs through the downregulation of Bcl2 expression, upregulation of BAX expression, and inhibition of the platelet-derived growth factor-β receptor (PDGF-βR)/p38 pathway. Zhang et al.⁷⁶⁾ suggested that, at a concentration of 2 µmol/L, SSd enhances the expression of the Fas death receptor, facilitates the cleavage of caspase-8, and subsequently activates the pro-apoptotic protein Bid. This sequence of events results in the release of mitochondrial cytochrome c and the activation of caspase-3, thereby culminating in hepatocyte apoptosis. It is noteworthy that this apoptotic mechanism does not significantly modulate the phosphatidylinositol 3-kinase (PI3K)/AKT, ERK, or STAT3 pathways, highlighting its specificity in mediating apoptosis via Fas and Bid activation. In in vivo studies, SSd elicited substantial hepatocyte apoptosis while downregulating the expression of the anti-apoptotic protein Bcl-2 and upregulating the pro-apoptotic protein BAX in mouse liver tissues, thereby corroborating the in vitro observations. Zhang et al.⁷⁷⁾ further elucidated the underlying mechanisms through metabolomic analyses: caspase inhibitors were found to mitigate SSd-induced hepatotoxicity, confirming that apoptosis plays a pivotal role in saikosaponin-mediated liver injury.

3.3. Induction of Hepatocyte Autophagy Injury

SSa also demonstrates hepatotoxicity, which is linked to autophagy. In HL-7702 cells, SSa reduces cell viability and enhances autophagic flux, as shown by increased LC3-II/LC3-I and Beclin-1 levels with concomitant p62 degradation. Mechanistically, SSa activates multiple ER stress pathways, including protein kinase RNA-like endoplasmic reticulum kinase/eukaryotic initiation factor 2 alpha/activating transcription factor 4 (ATF4)/C/EBP homologous protein (CHOP) inositol-requiring enzyme 1 (IRE1)–TNF receptor-associated factor 2 (TRAF2), activating transcription factor 6 (ATF6), and AMP-activated protein kinase (AMPK)/mTOR signaling, collectively driving excessive autophagy and cell injury. Inhibitors of autophagy or ER stress mitigate these effects and improve cell survival, confirming the causal role of these pathways. Consistent findings in vivo demonstrate that oral SSa (150 to 300 mg/kg) induces liver injury in mice, elevating LC3-II, 78-kDa glucose-regulated protein (GRP78), and CHOP while reducing p62. These results indicate that SSa-induced hepatotoxicity arises from ER stress–dependent overactivation of autophagy, warranting careful dosing and mechanistic evaluation in future applications.⁷⁸⁾ Recent studies have discovered that SSd disrupts protective autophagy by targeting the gamma-aminobutyric acid receptor-associated protein (GABARAP)-soluble NSF attachment protein receptor (SNARE) complex.⁷⁹⁾

In summary, the hepatotoxicity of SSs involves oxidative stress, apoptosis, autophagy, and other mechanisms (Table 2). However, the following three aspects warrant attention.

Table 2. Hepatotoxic Effects and Molecular Mechanisms of Saikosaponins

SSs	Experimental model	Dosage	Key findings	Ref.
SSd	HepaRG cells	0.5–10 µmol/L	CYP3A4↓	74)
SSd	LO2 cells	0.4–2 µM	PDGF-βR/p38↓	75)
SSd	LO2 cells　 ICR mice	2 µM.　 300 mg/kg (i.g.)	Fas↑, cleaved caspase-8↑, cleaved caspase-3↑, Bcl-2↓, Bax ↑	76)
SSd	ICR mice	25 mg/kg (i.p.)	cleaved caspase-1↑, IL-1β↑, IL-18↑, TNF-α↑	77)
SSd	MPHs　 HepG2 cells　 C57BL/6J mice	0.3125 µM　 2.5 µM　 25 and 50 mg/kg (i.g.)	Targeting the GABARAP-SNARE Complex to Disrupt Protective Autophagy	79)
SSa	LO2 cells　 ICR mice	5–10 µL　 150 and 300 mg/kg (i.g.)	PERK/eIF2α/ATF4/CHOP↑, IRE1/p-IRE1/TRAF2↑, ATF6↑, AMPK/mTOR↑, p62↓, GRP78, CHOP, and LC3-II↑	78)

Bax Bcl-2-associated X protein, GPER1 G protein-coupled estrogen receptor 1, i.g. intragastric administration, i.p. intraperitoneal injection, LC3-II light chain 3 II, PDGF platelet-derived growth factor, SS Saikosaponin, SSs Saikosaponins.

First, SSd is the most hepatotoxic SSs, inducing significant hepatocyte apoptosis via Fas-mediated pathways, while SSa and SSc have lower cytotoxicity due to structural differences.^12,79,80) Animal studies confirm that high doses of SSd cause severe liver injury, whereas SSa and SSc lead to milder effects.⁷³⁾ SSd also upregulates CYP2E1, increasing oxidative stress, and exhibits higher bioavailability and slower clearance, enhancing liver injury risk.^36,73) These findings highlight the need to differentiate SSs subclasses for safer therapeutic applications.

Second, SSs demonstrate paradoxical roles in the modulation of apoptosis, oxidative stress, and liver fibrosis, offering protective benefits at lower concentrations while potentially eliciting hepatotoxicity at elevated levels. Toxicological investigations suggest that excessive consumption of SSs or Bupleurum formulations may lead to either acute or chronic hepatic injury, characterized by the upregulation of CYP enzymes (e.g., CYP2E1) and increased oxidative stress.¹²⁾ Studies have indicated that pronounced acute liver damage occurs in rats exclusively at doses surpassing 12.957 mg/kg, which is approximately eightfold the clinically established daily safe dosage.⁷³⁾

Finally, the hepatotoxicity risk of SSs may be influenced by other drugs, triggering drug interactions that subsequently affect liver safety, and altering the metabolic and toxicological profiles of SSs in polypharmacy regimens. Zhao et al.⁵⁶⁾ demonstrated that SSb2 inhibits the activity of hepatic drug transporters MRP1, MRP2, and Pgp, potentially increasing hepatic accumulation and hepatotoxicity risk of co-administered drugs. Fan et al.⁷⁹⁾ elucidated the mechanism underlying the hepatotoxicity risk associated with the combined use of SSd and APAP. SSd exacerbates liver injury by inhibiting the fusion of autophagosomes with lysosomes, thereby impairing APAP-induced protective autophagy. Mechanistically, SSd directly binds to GABARAP protein, disrupting the formation of SNARE complexes and consequently obstructing autophagic flux. Notably, liposome encapsulation improves drug stability and bioavailability while significantly reducing SSd toxicity.^36,81)

4. DISCUSSION

Research on SSs, particularly SSd, has revealed both promising hepatoprotective activities and notable hepatotoxic risks, resulting in ongoing controversy and several limitations in the field. A major point of debate concerns the dual roles of SSs in liver pathology: although they exhibit anti-inflammatory, anti-fibrotic, and anti-tumor properties, they may also induce acute or chronic liver injury, especially at high doses or with prolonged exposure. Li et al.¹²⁾ summarized that the contradictory effects of SSs on apoptosis, oxidative stress, and fibrosis complicate the interpretation of their overall clinical impact, underscoring the need for detailed mechanistic and dose–response investigations.

Notably, the dual bioactivities of SSs are governed by strict dose dependency and microenvironmental specificity, wherein protective and toxicity effects manifest exclusively within the same cell type or animal model rather than across distinct cellular or physiological contexts. For instance, in Institute of Cancer Research (ICR) mice, low-dose SSd (1 to 2 mg/kg, i.p.) confers hepatoprotection by suppressing mitochondrial ROS-mediated oxidative stress and NLRP3 inflammasome activation, whereas a high dose (25 mg/kg, i.p.) triggers caspase-1-mediated inflammatory cell death.^61,77) Furthermore, half-maximal IC₅₀ values depend on factors such as cell type and SS subtype^47,75,79): SSd displays cell type-specific potency (LO2 cells: 2.14 µM; HepG2 cells: 2.63 µM; Hep3B cells: 4.26 µM) and compound-specific selectivity within HepG2 cells (SSd: 2.90 µM; SSa: 6.00 µM; SSb₂: 23.09 µM), confirming that operational definitions of “low” versus “high” dose are intrinsically determined by the sensitivity threshold of a given cell type or animal model toward a specific SSs. This paradigm underscores that SSs bioactivity is exquisitely modulated by the cellular microenvironment—a foundational principle for the rational design of targeted SSs-based therapeutics with minimized off-target hepatotoxicity.

Toxicological concerns are amplified by the complex metabolic processes of SSs and their metabolites, many of which remain insufficiently characterized. DILI caused by herbal components exhibits multifactorial characteristics, involving oxidative stress, apoptosis, and autophagy, which poses challenges for prediction and modeling.^82–84) These complexities are reflected in inconsistent reporting of herbal hepatotoxicity cases. Agarwal et al.⁸⁵⁾ noted that many case reports lack essential diagnostic data, limiting accurate causality assessment and clinical interpretation.

Limitations in current preclinical models further hinder progress. Conventional animal models and in vitro systems inadequately recapitulate human liver physiology and idiosyncratic toxicity. Although primary human hepatocytes and advanced 3D cultures improve translational relevance, species differences and the absence of standardized protocols remain significant obstacles.^86,87) Moreover, studies by Ortega-Vallbona and Li et al.^88,89) demonstrated that minor structural modifications can markedly alter hepatotoxic potential, highlighting the need for refined structure–activity relationship analyses.

Pharmacokinetic challenges also restrict clinical development. Zhou et al.⁵⁷⁾ reported that SSd exhibits poor oral bioavailability and interacts with CYP enzymes and P-glycoprotein, potentially modifying the pharmacokinetics of co-administered drugs and contributing to toxicity. Among the proposed mitigation strategies, liposomal encapsulation effectively reduced SSd-induced cytotoxicity and improved therapeutic efficacy in vivo, although these formulations require further clinical validation.³⁶⁾

Another major limitation is the scarcity of high-quality clinical trials evaluating the safety and efficacy of SSs in liver diseases. Most available data originate from preclinical research or anecdotal case reports, while heterogeneity in dosages, formulations, and diagnostic criteria further complicates evidence synthesis. Calitz et al.⁹⁰⁾ highlighted additional concerns related to variability in herbal product quality, contamination, and the lack of standardized causality assessment methods.

Despite these challenges, SSs—particularly SSd and SSa—exhibit substantial therapeutic potential. Their anti-inflammatory, anti-fibrotic, and anti-tumor activities suggest valuable roles across various liver disease contexts. Natural compounds, including SSs, are being explored for their ability to mitigate chemotherapy-induced liver injury, with evidence showing reduced biochemical markers and modulated inflammatory responses in hepatotoxicity models.⁹¹⁾ In cancer therapy, SSs may also act as chemosensitizers. Song et al.⁵⁵⁾ demonstrated that SSa enhanced gemcitabine sensitivity in intrahepatic cholangiocarcinoma via modulation of the p-AKT/BCL-6/ABCA1 axis, suggesting synergistic application in hepatobiliary malignancies. Likewise, Lv et al.⁹²⁾ found that SSd, particularly when combined with neuropilin-1 knockdown, disrupted lipid transport and phospholipid metabolism in HepG2 cells, supporting its anti-hepatoma efficacy.

Future research should prioritize elucidating the molecular pathways that underlie the hepatoprotective and hepatotoxic effects of SSs. Wang et al.⁹³⁾ emphasized the importance of understanding how SSs regulate NF-κB and MAPK signaling and interact with the gut microbiome, which may ultimately shape systemic immunity and liver health. Advances in omics and computational modeling offer further opportunities for mechanistic discovery and toxicity prediction. Umbaugh and Jaeschke⁹⁴⁾ highlighted the utility of integrating multi-omics with machine learning to identify DILI biomarkers, while Przybylak and Cronin⁹⁵⁾ discussed in silico frameworks incorporating chemical and toxicogenomic profiles for hepatotoxicity prediction.

Improving preclinical models remains a critical priority. Standardized protocols for liver injury models, as recommended by Ezhilarasan et al.,⁹⁶⁾ will improve reproducibility and translational relevance. Human pluripotent stem cell–derived hepatocytes and 3D co-culture systems reviewed by Tuschl et al.⁹⁷⁾ may better capture human-specific physiology and idiosyncratic toxicity.

Pharmacokinetic and formulation optimization should also be advanced through strategies such as liposomal encapsulation, nanoparticle delivery, and structural modification.^36,98) Moreover, evaluating drug–drug interactions—particularly those involving CYP enzymes and transporters—is essential for preventing adverse clinical events.⁵⁷⁾

Advancing clinical development will require well-designed randomized controlled trials using standardized SSs formulations. Lin and Li⁹⁹⁾ emphasized the need for robust regulatory oversight and innovative trial methodologies in liver therapeutics, which may guide global efforts. Improved frameworks for evaluating herbal hepatotoxicity, as recommended by Calitz et al.,⁹⁰⁾ will strengthen safety assessment.

Combination therapies also represent a promising direction. Early studies suggest synergistic effects when SSs are combined with chemotherapeutic agents or metabolic drugs such as metformin and statins,^55,100,101) warranting further exploration in clinical settings.

In summary, SSs research faces significant challenges—including conflicting hepatoprotective versus hepatotoxic effects, incomplete mechanistic understanding, limited preclinical models, complex pharmacokinetics, and insufficient clinical evidence. Nonetheless, the prospects for clinical translation remain strong, supported by expanding preclinical data and advances in delivery technologies. Moving forward, rigorous clinical trials, standardized formulations, and targeted mechanistic studies will be essential to close existing knowledge gaps and enable the safe and effective application of SSs in liver disease therapy.

5. CONCLUSION

SSs represent a promising yet paradoxical class of bioactive compounds in the treatment of liver diseases. A substantial body of preclinical evidence demonstrates that SSs exert broad-spectrum hepatoprotective effects, mediated through the modulation of key signaling pathways such as NF-κB, STAT3, LXRα, ERβ, and COX-2, thereby producing anti-inflammatory, anti-fibrotic, and anti-tumor activities. Nonetheless, these beneficial effects coexist with dose-dependent hepatotoxic potential, particularly for SSd, which can induce oxidative stress, apoptosis, and autophagy-related liver injury. The dual nature of SSs underscores the critical importance of dosage optimization, mechanistic clarification, and safety evaluation.

To advance clinical translation, future efforts should integrate systems pharmacology, omics-based toxicology, and advanced in vitro and in vivo liver models to elucidate precise mechanisms of action and predict toxicity. Novel drug delivery systems, including liposomal and nanoparticle formulations, hold promise for enhancing bioavailability and minimizing adverse effects. Furthermore, combination therapy strategies involving SSs and established hepatoprotective or chemotherapeutic agents may yield synergistic benefits. Ultimately, rigorously designed clinical trials using standardized preparations are essential to validate efficacy, define safety profiles, and support regulatory approval. With continued interdisciplinary research, SSs may evolve from traditional medicinal components into scientifically validated therapeutics for liver diseases.

DECLARATIONS

Funding

Guangzhou Health Science and Technology Project (20251A011020) and Guangzhou Science and Technology Plan Project (2024A03J0659).

Author Contributions

All authors contributed to designing, writing, and revising the manuscript.

Conflict of Interest

The authors declare no conflict of interest.

Data Availability

All data are included in the manuscript.

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