Cerebral ischemia/reperfusion (I/R) injury is commonly associated with high rates of disability and mortality due to its complex pathological mechanisms. CX, a traditional medicine, is widely used in the treatment of ischemic stroke. Several metabolites of CX have demonstrated efficacy in managing cerebral ischemia by modulating oxidative stress, protecting blood vessels, and regulating inflammation. For instance, the National Medical Products Administration (NMPA) recognized butylphthalide as a therapeutic agent for ischemic stroke in 2005 (Zou et al., 2018). TMP inhibits platelet adhesion and the release of inflammatory factors by suppressing the P38 mitogen-activated protein kinase (MAPK) and nuclear factor kappa-B (NF-κB) signaling pathways, thereby protecting microvascular endothelial cells in the brain (Zhang H. et al., 2020). Ligustilide activates adenosine 5‘-monophosphate (AMP)-activated protein kinase (AMPK) signaling and PTEN induced putative kinase 1 (PINK1)/Parkin expression, which helps alleviate ischemic stroke damage (Mao et al., 2022; Wu et al., 2022). In a mouse model of induced intracerebral hemorrhage, ligustilide and senkyunolide H were shown to exert potent neuroprotective effects against hemorrhagic stroke by inhibiting the Paired Related Homeobox 1 (Prx1)/Toll-like receptor 4 (TLR4)/NF-κB pathway, and subsequent immune and neuroinflammatory damage (Han et al., 2018). In a subacute ischemic rat model, TMP was found to inhibit the overexpression of aquaporin 4 (AQP4) and connexin 43 (Cx43) in astrocytes by altering A1-A2 reactivity. TMP also activates the fibroblast growth factor (FGF) 2/phosphoinositide 3-kinase (PI3K)/Akt pathway, which may contribute to its promotion of neurovascular repair following ischemic stroke (Feng X. F. et al., 2023). Additional studies have reported that CX and Binpian [Cinnamomum camphora(L.)Presl] exhibit significant synergistic effects in promoting neurogenesis and reducing astrocyte generation, with CX playing a central role and Binpian enhancing its effects. Binpian is frequently combined with CX to improve the treatment of ischemic stroke (Song et al., 2023; Zhang X. et al., 2024).

Senkyunolide A and Z-ligustilide, two isolated metabolites, demonstrate inhibitory effects on neuroinflammation (Or et al., 2011). In microsphere-embolized (ME) rats, CX promotes neurogenesis and reduces inflammatory factors, thereby protecting neurons from cerebral ischemia (Wang et al., 2020). TMP has been shown to decrease the levels of inflammatory factors, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and matrix metalloproteinase (MMP)-9, in ischemic brain tissue, exerting neuroprotective effects in mice subjected to I/R injury. This neuroprotective effect may be mediated by the upregulation of monocyte chemoattractant protein-induced protein 1 (MCPIP1), as the neuroprotection and anti-inflammatory effect were inhibited upon knockdown of MCPIP1 using specific small interfering RNA (siRNA) (Jin et al., 2021). TMP also protects retinal ganglion cells by inhibiting apoptosis and autophagy via the PI3K/Akt/mammalian target of rapamycin (mTOR) pathway. Additionally, Senkyunolide I has been found to protect mouse neuroblastoma cells from glutamate-induced injury by downregulating the c-Jun N-terminal kinase (c-JUN)/cysteinyl aspartate specific proteinase 3 (caspase-3) pathway and attenuating apoptosis (Wang M. et al., 2021). Furthermore, ligustilide exerts protective effects against oxygen-glucose deprivation/reperfusion (OGD/R) injury in PC12 cells by inhibiting apoptosis through the mitochondrial pathway and promoting autophagy via the Liver kinase B1 (LKB1)-AMPK-mTOR signaling pathway (Zhao et al., 2020). Ma et al. (2024) found that QBT, a novel phthalein metabolite, significantly reduced cognitive dysfunction and neuronal damage in rats with vascular dementia (VaD). It also inhibited VaD-induced hyperactivation of microglia and astrocytes, along with associated inflammatory responses. Moreover, QBT exerts anti-inflammatory effects by inhibiting the chemokine C-X-C motif ligand 12 (CXCL12)/chemokine C-X-C motif receptor 4 (CXCR4) axis and its downstream Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) and PI3K/Akt/NF-κB pathways, thereby attenuating neuroinflammatory responses both in vivo and in vitro.

Alzheimer’s disease (AD) is the most common neurodegenerative disorder, leading to various functional impairments, including memory loss, dementia, and aphasia. It is the third leading cause of disability and death in the elderly, following cardiovascular diseases and malignant tumors, and is considered a major societal burden (Jeremic et al., 2021). Several hypotheses have been proposed regarding the pathogenesis of AD, with the most widely accepted mechanisms involving the excessive accumulation of beta-amyloid (Aβ) outside neurons and the aggregation of hyperphosphorylated tau protein within neurons, resulting in the formation of neurofibrillary tangles (NFTs) (Du et al., 2018; Scheltens et al., 2021). Despite extensive efforts to develop therapeutic strategies for AD, only a few drugs have been approved for treatment, all of which merely provide symptomatic relief and are associated with significant side effects. Effective cures or preventive treatments remain scarce.

In the context of tau pathology, CX has been reported to target 70 key pathways associated with tau-related pathological mechanisms, including interactions with astrocytes, endothelial cells, and microglia. This action effectively reverses tau hyperphosphorylation and promotes its degradation through the lysosomal pathway, thereby mitigating tau-mediated intracellular transport impairment, axonal and synaptic damage, and neuronal death. The primary active metabolites identified in this process include neocnidilide, ferulic acid, Z-ligustilide, and butylidene phthalide, which have demonstrated neuroprotective effects against AD. In contrast, TMP, a widely used therapeutic agent for I/R-induced brain injury, has been associated with only two tau-related targets, suggesting limited therapeutic relevance in tau pathology-based AD (Zeng et al., 2022). However, compared to the control group, the TMP-related group exhibited significant improvements in learning and memory abilities in transgenic AD mice, along with reduced levels of Aβ and tau phosphorylation in the brain (Huang et al., 2021). These effects may be attributed to TMP’s ability to regulate mitochondrial function, maintain synaptic integrity and cytoskeletal stability, and modulate proteins associated with adenosine triphosphate (ATP) and guanosine triphosphate (GTP) binding. Another potential mechanism underlying TMP’s anti-AD’s effects involves the downregulation of Cullin 4B (CUL4B) in the brain tissue of AD mice, which inhibits the ubiquitination of somatostatin receptor 4 (SSRT4) and upregulates SSRT4 protein levels. This mechanism has been confirmed in mice with CUL4B overexpression or SSRT4 silencing (Weng et al., 2021). Additionally, TMP may partially restore cholinergic neuronal function in streptozotocin (STZ)-induced AD rats by inhibiting glycogen synthase kinase (GSK)-3β activity, thereby improving learning and memory deficits (Lu F. et al., 2017).

One study demonstrated that QBT exhibits strong biological activity against cerebrovascular diseases. In VaD rats, QBT reversed aberrant changes associated with ferroptosis and pyroptosis by upregulating the nuclear factor erythroid 2-related factor 2 (Nrf2)/cystine-glutamate antiporter (xCT)/glutathione peroxidase 4 (GPX4) pathway, downregulating the nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3)/cysteine-requiring aspartate protease-1 (Caspase-1)/gasdermin D (GSDMD) pathway, and inhibiting both ferroptosis and pyroptosis in neuronal cells. These actions exerted neuroprotective effects, significantly reducing neuronal damage and cognitive dysfunction in VaD rats (Feng et al., 2024). Furthermore, the methanol extract of CX effectively mitigated cognitive impairment caused by bilateral common carotid artery stenosis, primarily through the inhibition of astrocytes and microglia (Lim et al., 2024).

Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterized by a gradual loss of motor function, with the primary pathological hallmarks being the sustained degeneration of dopaminergic neurons in the substantia nigra and the intracellular accumulation of α-synuclein in the form of Lewy bodies and Lewy neurites (Kouli et al., 2018). CX may play a role in PD biology, as 53 metabolites in CX have been found to affect the biological compartments of human neuronal oxidative stress (hNOS) cells derived from PD patients. These metabolites may interfere with the biological functions of cellular organelles, including early endosomes, lysosomes, and autophagosomes. Additionally, TMP downregulates neuroinflammation markers such as NF-κB, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX2) (Michel et al., 2017). Canhui and Mengchan (2023) found that ligustilide improved the survival rate of PD cell models, reduced cell cycle arrest in the G0/G1 phase, and inhibited apoptosis, primarily by suppressing the activation of the p38 MAPK/c-JUN signaling pathway.

The antimigraine effects of total alkaloids in CX have been confirmed to be mediated by the upregulation of 5-hydroxytryptamine (5-HT) and its metabolite 5-hydroxyindole acetic acid (5-HIAA), achieved through increased expression of the 5-Hydroxytryptamine Receptor 1B (5-HT1B) and suppression of c-Jun (Pu et al., 2019b). Furthermore, TMP selectively inhibits c-Jun phosphorylation and reduces the expression of MMP-2/9 and pro-inflammatory cytokines secreted by astrocytes, thereby alleviating neuropathic pain caused by chronic compression injury (CCI). TMP also inhibits the activation of the NF-κB pathway in microglia by upregulating the expression of silent information regulator 1 (SIRT-1), thereby reducing neuroinflammatory responses and alleviating migraine (Chen et al., 2024).

Huntington’s disease (HD) is a neurodegenerative disorder with an autosomal dominant inheritance pattern, primarily characterized by motor dysfunction, ataxia, and cognitive decline. 3-Nitropropionic acid (3-NP) is commonly used to induce HD-like symptoms in animal models. Treatment with TMP significantly improves behavioral outcomes in 3-NP-induced HD rats, with biochemical analyses indicating a marked reduction in oxidative stress parameters (Danduga et al., 2018).

Myocardial ischemia improvement is one of the most significant pharmacological actions of CX and its most widely applied use in clinical practice. Research indicates that CX exerts anti-inflammatory, antioxidative, and anti-apoptotic effects on cardiomyocytes subjected to I/R injury, while also promoting coronary blood flow and myocardial metabolism, thereby limiting the extent of myocardial infarction (Zheng et al., 2018). Additionally, studies have shown that CX protects against acute myocardial ischemia induced by isoproterenol through activation of the PI3K/Akt/GSK-3 signaling pathway (Yang et al., 2019).

Wang et al. (2024) demonstrated that TMP intervention reduced cardiomyocyte apoptosis induced by OGD/R and I/R, enhanced cellular viability and autophagy, and improved myocardial tissue necrosis in I/R mice in a dose-dependent manner. Ligustilide has been shown to attenuate myocardial injury caused by inflammation, reduce fibrosis and hypertrophy of myocardial cells, and inhibit ventricular dilation following myocardial infarction. Furthermore, ligustilide provides protective effects in familial dilated cardiomyopathy. When combined with other Chinese herbal medicines, ligustilide can enhance therapeutic efficacy synergistically; for example, its combination with berberine has demonstrated more effective antiplatelet and anti-inflammatory effects in rats with acute myocardial infarction (Zhang et al., 2016).

In addition to inflammation and oxidative stress, autophagic dysfunction plays a significant role in various cardiovascular diseases. Studies have demonstrated that the lactone metabolite of ligustilide can activate the PI3K/Akt/mTOR signaling pathway, restore autophagic flux, and exert cardioprotective effects (Wang et al., 2018). Moreover, CX protects the myocardium from I/R injury by inhibiting Beclin-1-related autophagic dysfunction and enhancing the translocation of B-cell lymphoma-2 (Bcl-2) through increased expression of 14-3-3γ(Zuo et al., 2019).

AS is characterized by the accumulation of lipids, complex carbohydrates, and/or thrombus formation, accompanied by a progressive fibrotic response and calcium deposition, ultimately leading to arterial thickening, hardening, and luminal narrowing (Libby, 2021). Zhang et al. (2024b) explored the mechanisms through which Zhizi (Gardenia jasminoides Ellis) and CX botanical drugs attenuate the progression of AS via Deoxyribonucleic Acid (DNA) methylation. The results indicate that both Zhizi and CX effectively slow AS progression by inhibiting MAPK/extracellular signal-regulated kinase (ERK)-mediated apoptosis through hypermethylation of the fibroblast growth factor receptor 3 (FGFR3) promoter region. Additionally, they alleviate AS by inhibiting the TNF/NF-κB axis and M1 macrophage polarization (Zhang et al., 2024c). Liu et al. (2024) demonstrated for the first time the potential of oral Z-ligustilide treatment to alleviate AS by reducing inflammation and enhancing intestinal barrier function. TMP exerts therapeutic effects on AS primarily through its anti-inflammatory, antioxidant, and lipid-lowering actions, effectively inhibiting lipid accumulation and enhancing intracellular cholesterol efflux. Key targets implicated in these mechanisms include peroxisome proliferator-activated receptors (PPAR), sterol regulatory element-binding proteins (SREBPs)/cleavage activating protein (SCAP), progesterone and adiponectin receptors 3, scavenger receptors (SR-A, CD36), ATP-binding cassette transporters (ABCA1 and ABCG1), and the PI3K/Akt and p38/MAPK signaling pathways (Chen et al., 2017; Duan et al., 2017).

In the management of hypertension, TMP has been reported to exert dual actions: it blocks the entry of extracellular Ca²⁺ through calcium channels and inhibits the release of Ca²⁺ stored within vascular smooth muscle cells (SMCs) (Ren et al., 2012). The Kcnma1 gene encodes the large conductance calcium-activated potassium channels (BKCa), which are critical for treating various cardiovascular, muscular, and nervous system disorders. Research has shown that TMP helps maintain BKCa β1 protein expression by inhibiting endoplasmic reticulum stress, restoring BKCa currents in porcine coronary artery SMCs, and improving BKCa-mediated vasodilation in coronary arteries (Sun et al., 2019; Szteyn and Singh, 2020).

Liguzinediol exhibits a dose-dependent positive inotropic effect in doxorubicin-induced heart failure mice, indicating its potential as a treatment for heart failure without causing adverse reactions such as arrhythmias or hypotension. This beneficial effect is primarily mediated by an increase in the bcl-2/Bcl-2 associated X protein (Bax) ratio and the suppression of caspase-3 expression and NK-κB activation (Li et al., 2014a). Sodium ferulate has been shown to mitigate myocardial hypertrophy induced by abdominal aorta coarctation by inhibiting protein kinase C beta (PKC-β) and activating the MAPK signaling pathways (Luo et al., 2019).

TMP also demonstrates inhibitory effects on thrombosis. It exerts its antithrombotic effects by reducing inflammatory cytokines and adhesion molecules, modulating MAPK signaling, attenuating oxidative stress, preventing apoptosis, inhibiting platelet aggregation, and suppressing the expression of fibrinogen and von Willebrand factor (Yang et al., 2023). In addition to TMP and ferulic acid, four other effective metabolites have been identified: neochlorogenic acid, 1-H-benzimidazole-2-amine, 3,8-dihydroxyacyl lactone, and CX triterpenes. These metabolites interact with the core amino acids of target proteins, effectively inhibiting blood clot formation.

Studies have found that CX adjunctive therapy can significantly improve the clinical symptoms of patients with chronic idiopathic pulmonary fibrosis (Shupei, 2010). The use of microemulsion formulations encapsulating the volatile oils of Danggui [Angelica sinensis (Oliv.) Diels] and CX has been shown to enhance their ability to alleviate acute lung injury (ALI) (Zhang et al., 2022). TMP is primarily regarded as an active medicinal metabolite of CX that targets the respiratory system. In vivo studies have demonstrated that TMP inhibits the proliferation of airway SMCs by reducing p-ERK 1/2 levels, a process associated with the MAPK signaling pathway (Li et al., 2018). Jiang et al. confirmed that TMP exhibits both anti-inflammatory and anti-apoptotic activities in macrophages, which have been validated in vivo and in vitro. These effects involve the TLR4/Tumor necrosis factor receptor-associated factor 6 (TRAF6)/NF-κB/NLRP3/caspase-1 and TLR4/cysteine-requiring aspartate protease-8 (caspase-8)/caspase-3 signaling pathways (Jiang R. et al., 2021). Furthermore, TMP has been shown to alleviate lung inflammation in an ovalbumin-induced asthma mouse model by downregulating C-C Motif Chemokine Receptor 1 (CCR1), STAT3, and p38 MAPK proteins, thereby inhibiting airway hyperresponsiveness and the release of inflammatory and chemotactic factors (Wei et al., 2016). These findings suggest that TMP may have potential in treating ALI and acute respiratory distress syndrome (ARDS) primarily through reducing inflammatory cell infiltration and lowering pro-inflammatory factor secretion in the alveoli. Furthermore, TMP has shown efficacy in alleviating ALI caused by sepsis, reducing inflammatory cell infiltration, and mitigating lipopolysaccharide-induced lung tissue damage. TMP significantly inhibits the Rac Family Small GTPase 1 (Rac1)/LIM Domain Kinase 1 (LIMK1)/Zonula occludens-1 (ZO-1)/occludin signaling pathway (Min et al., 2022).

Ferulic acid demonstrates anti-hepatic stellate cell (HSC) activity by inhibiting focal adhesion kinase (FAK) and blocking the Smad and ERK1/2 signaling pathways, highlighting its potential in treating liver fibrosis (Xu et al., 2015). In a rat model of thioacetamide-induced liver fibrosis, butylidene phthalide exhibited significant anti-fibrotic effects by inhibiting epithelial-mesenchymal transition, reducing inflammatory responses, and promoting hepatocyte proliferation (Chuang et al., 2016). Additionally, CX combined with Danggui has been shown to prevent liver fibrosis (Wu et al., 2021). TMP has been reported to effectively attenuate hepatic I/R injury by inhibiting neutrophil extracellular trap formation and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Liu et al., 2021). In the study by Lu et al., TMP was also found to confer hepatoprotective effects and improve hepatic steatosis via Nrf2 activation (Lu C. et al., 2017). Furthermore, Lu et al. (2023) reported that the organic acids in CX can alleviate liver injury through multiple signaling pathways, primarily involving the positive regulation of nitric oxide (NO) biosynthesis, Toll-like receptor signaling, NOD-like receptor signaling, and TNF signaling pathways. Additionally, the phthalate ester extract from CX exhibits stronger anti-cholestatic activity, both in vivo and in vitro, compared to other CX extracts. Its protective effect on bile duct cells and HSCs is mediated by reducing the transcription and release of plasminogen activator inhibitor-1 (PAI-1) and fibronectin through lysine acetyltransferase 2A (KAT2A)/histone 3 lysine 9 acetylation (H3K9ac) (Li Y. et al., 2023).

Studies have shown that CX and Dahuang(Rheum officinale Baill.) may inhibit renal tubular epithelial cell apoptosis and improve acute kidney injury (AKI) and renal fibrosis by suppressing the p38 MAPK/p53 signaling pathway (Li J. et al., 2023). Xia et al. (2024) found that ligustilide alleviates oxidative stress during renal I/R injury by maintaining Sirt3-dependent mitochondrial homeostasis. Qi et al. (2024) identified potential effective metabolites of CX in renal protection, revealing that phthalides improve hyperglycemia-induced renal dysfunction by enhancing Nrf2 activation, reducing collagen deposition, and alleviating inflammation and oxidative stress. Levistolide A mitigates AKI through its antioxidant properties and modulation of the TLR4/NF-κB signaling pathway (Shi J. et al., 2024). TMP exhibits anti-apoptotic and therapeutic effects in diabetic nephropathy (DN) rats by activating the Akt signaling pathway and reducing oxidative stress (Rai et al., 2019). Its renal protective effects include attenuation of renal pathological changes, reduction in blood urea nitrogen, serum creatinine, 24-h urinary albumin, and glycated hemoglobin (HbA1c) levels, as well as an improvement in creatinine clearance (Zhuang et al., 2020). Additionally, TMP plays a significant protective role in AKI, primarily by downregulating the expression of nucleotide-binding oligomerization domain 2 (NOD2), which reduces renal cell apoptosis in a rat ischemia-reperfusion injury model (Jiang G. et al., 2021). TMP has been applied clinically to mitigate AKI. Senkyunolide I alleviates renal I/R injury by inhibiting oxidative stress, endoplasmic reticulum stress, and apoptosis (Zhu et al., 2022).

Tumor metastasis plays a critical role in the progression of malignant tumors, inhibiting autophagy and apoptosis in cancer cells, thereby promoting uncontrolled cell division and proliferation. CX, a natural plant extract, contains various metabolites with multi-targeted antitumor activities, including the promotion of tumor cell apoptosis and the inhibition of metastasis (Wang K. et al., 2021). Perlolyrine has been identified as a metabolite with antiproliferative effects on human gastric cancer cell lines (Lee et al., 2016); metabolites CX0, CX1, and CX2 exhibit inhibitory effects on the growth of HepG2, SMMC7721, A549, and HCT-116 cells (Hu et al., 2016a); metabolites CXP-1a and CXP-3a stimulate macrophages to produce NO, TNF-α, IL-6, and IL-12, while inhibiting the growth of HepG2 and Hep3B cells through immune modulation by blocking the cell cycle in the G0/G1 phase, thus promoting cancer cell apoptosis (Zhong et al., 2021). Jung et al. (2025) elucidated the molecular mechanisms underlying TMP’s anticancer effects: TMP induces apoptosis in HCCLM3 and Hep3B cells by activating key apoptosis factors and inhibiting proteins associated with cell survival and angiogenesis. It enhances autophagy by promoting the formation of autophagosomes and stimulating autophagy-related proteins, while upregulating SHP-1 to inhibit the activation of the STAT3 signaling pathway, thereby suppressing tumorigenesis and activating cell death pathways. TMP significantly inhibits tumor growth and triggers apoptosis and autophagy in tumor tissues.