Leonurine: A Multifaceted Bioactive Alkaloid-From Pharmacological Mechanisms to Clinical Translation: A Narrative Review.

Zhouyue Gu: conceptualization, visualization, project administration, writing – review and editing, writing – original draft.

The author received no specific funding for this work.

Hyperlipidemia represents a significant risk factor for cardio‐cerebrovascular diseases, creating an urgent need for effective and well‐tolerated therapeutic options [ 101 ]. Preclinical investigations have demonstrated LEO's potent lipid‐modulating capabilities, with three separate hyperlipidemia model studies confirming its total cholesterol and triglyceride reduction efficacy comparable or superior to statins while exhibiting significantly fewer adverse effects [ 102 ]. This promising therapeutic profile has propelled LEO into clinical development, with multiple Phase I trials initiated since 2018 to evaluate its tolerability, safety, pharmacokinetics, and food interaction profiles through both single and multiple administration regimens. While specific trial outcomes remain unpublished at this stage, these clinical studies represent a crucial translational milestone in LEO's development pathway from laboratory research to potential clinical application The ongoing trials systematically assess critical parameters including dose–response relationships, bioavailability, and safety margins that will inform subsequent Phase II efficacy studies in patient populations (Table  3 ). Clinical trial information of LEO.

Leonurine, a bioactive alkaloid derived from Leonurus species, exhibits a wide range of pharmacological effects, including anti‐inflammatory, antioxidant, antiapoptotic, and antitumor activities. It has shown therapeutic promise in gynecological disorders, cardiovascular diseases, neurological conditions, and various cancers. Despite robust preclinical evidence, its clinical translation remains limited by poor oral bioavailability, incomplete understanding of molecular targets, and a lack of human clinical data. These limitations highlight the need for structural optimization, advanced drug delivery strategies, and comprehensive mechanistic studies. Moreover, rigorously designed clinical trials are essential to establish its safety and efficacy in humans. Addressing these gaps could position leonurine as a valuable natural compound in modern drug development. While adverse effects have been reported at high or prolonged doses, particularly when using crude ethanolic extracts, the purified form of leonurine has shown a favorable safety profile in multiple preclinical studies at therapeutic doses (10–30 mg/kg). Histopathological evaluations in rodents and nonhuman primates have revealed no significant toxicity at these levels. To ensure safe clinical translation, we recommend a phased, dose‐escalation trial design beginning with conservative human equivalent doses extrapolated from preclinical NOAEL data. Such trials should incorporate strict monitoring of hepatic, renal, and neurological parameters. This approach is consistent with standard protocols in early‐phase drug development, even for compounds with dose‐dependent toxicity.

Leonurus , a genus within the Lamiaceae family, includes Leonurus japonicus , an annual or biennial medicinal plant that has extensive use in traditional medicine. It is commonly employed to manage gynecological conditions such as menstrual irregularities, dysmenorrhea, amenorrhea, postpartum complications (including hemorrhage and abdominal pain), as well as urinary disorders like edema, dysuria, and hematuria. Additionally, it is applied in treating traumatic injuries and dermatological conditions such as carbuncles and toxic swellings. Its therapeutic significance is recognized [ 1 ] in the European Pharmacopoeia's seventh edition [ 2 ]. Over 280 secondary metabolites, including alkaloids, terpenoids, flavonoids, phenylpropanoids, polysaccharides, and volatile oils, have been identified in Leonurus [ 3 ]. Among these, leonurine (LEO) (Figure  1A ) is a distinctive alkaloid found in L. japonicus , present in fresh herb at concentrations of 0.02%–0.12%, and serves as a primary bioactive constituent [ 4 ]. Structure, metabolism, and therapeutic action. (A) Chemical structure of LEO. (B) Therapeutic mechanism in endometriosis (EMS), highlighting inhibition of aromatase–estrogen signaling and MST1‐mediated anti‐inflammatory effects. (C) Metabolic pathways showing key enzymes (CYP2D6, CYP1A2, CYP3A4, UGT1A1) and formation of major metabolites. CYP = Cytochrome P450, EMS = endometriosis, LEO = leonurine, MST1 = mammalian STE20‐like protein kinase 1, UGT = UDP‐glucuronosyltransferase. In recent years, there has been a surge of scientific interest in LEO, not only to validate its traditional uses but also to uncover its broader therapeutic potential through modern pharmacological approaches [ 2 , 5 ]. This shift reflects a growing trend in drug discovery that seeks to bridge traditional medicine with mechanistic understanding and clinical applicability. Therefore, this review aims to consolidate current knowledge of LEO's extraction, synthesis, pharmacokinetics, and multifaceted pharmacological activities, while highlighting its promise for clinical translation in diverse disease areas. Despite these promising preclinical findings, several translational hurdles remain unresolved. LEO exhibits extremely low oral bioavailability due to extensive first‐pass metabolism, and its precise molecular targets in humans are still being elucidated. Furthermore, human clinical trials are scarce, leaving its therapeutic efficacy and safety largely unconfirmed in real‐world settings. Therefore, this review aims to consolidate current knowledge of LEO's extraction, synthesis, pharmacokinetics, and multifaceted pharmacological activities, while highlighting its promise for clinical translation in diverse disease areas. Research has demonstrated that LEO exhibits diverse pharmacological properties, such as stimulating uterine contractions, mitigating oxidative stress and inflammation (Figure  1B ), modulating apoptosis, inhibiting tumor growth, and promoting angiogenesis. Additionally, it provides safeguarding benefits for various organ systems, such as the uterus, heart, blood vessels, nervous system, liver, kidneys, skin, bones, and even against tumors. Latest studies have particularly focused on its cardioprotective, cerebrovascular, and neuroprotective roles [ 5 ]. This review consolidates current knowledge of LEO's extraction techniques, synthetic and biosynthetic pathways, pharmacokinetics (Figure  1C ), pharmacological actions across diseases, toxicological profile, and clinical applications. The analysis draws from literature retrieved from PubMed, Web of Science, and China National Knowledge Infrastructure, primarily spanning 2018–2023, with selective inclusion of earlier pivotal studies for contextual depth. The extraction and synthesis of LEO trace back to its initial isolation from Leonurus in 1930, followed by refined methodologies such as acidic methanol extraction and column chromatography [ 6 ], with structural confirmation via IR, NMR, and MS [ 7 ]. Advanced techniques like high‐performance liquid chromatography [ 8 ], high‐speed countercurrent chromatography [ 9 ], and acidic ionic liquid ultrasonic‐assisted extraction [ 10 ] have since enhanced efficiency. Chemically, LEO has been synthesized via guanidine‐butanol condensation with syringic acid [ 11 ] or through multi‐step routes using trimethoxybenzoic acid [ 12 ]. Biosynthetically, multi‐omics studies reveal LEO's production in L. japonicus via arginine‐derived guanbutol and UDP‐glucosylated syringic acid, catalyzed by serine carboxypeptidase‐like acyltransferases in vacuoles, with UGT and SCPL gene expansions driving its species‐specific accumulation [ 13 ]. Figure  2 presents a schematic overview of the review's organizational structure, beginning with leonurine's pharmacokinetics and metabolism, followed by its pharmacological activities, toxicity evaluation, structure–activity relationships, clinical relevance, and future research directions. Schematic overview of the review structure on leonurine (LEO), highlighting its pharmacokinetics, pharmacological activities, toxicity, structure–activity relationships, clinical relevance, and future directions.

The lead author Zhouyue Gu affirms that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned (and, if relevant, registered) have been explained.

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

This comprehensive review has systematically examined Leonurine's extraction methodologies, synthetic pathways, biosynthetic mechanisms, pharmacokinetic properties, diverse pharmacological effects, toxicological profile, and current clinical trial progress. As a distinctive alkaloid exclusive to the Leonurus genus, LEO has demonstrated substantial therapeutic potential across multiple disease domains. The scientific community has made remarkable advancements in understanding LEO's pharmacokinetic behavior, pharmacological mechanisms, and safety parameters, while simultaneously achieving significant progress in biosynthesis techniques and pharmaceutical preparation technologies ‐ critical foundations for industrial‐scale production. Despite these achievements, several key challenges demand urgent attention. The limited botanical sources and growing therapeutic demand necessitate development of more cost‐effective and efficient synthetic routes alongside optimized extraction protocols. While preliminary pharmacokinetic studies in animal models have established baseline parameters, the ongoing clinical pharmacokinetic trials represent only initial steps toward comprehensive understanding. The compound's pharmacological limitations ‐ particularly its low bioavailability, extensive first‐pass metabolism, and enterohepatic recirculation ‐ may potentially be addressed through targeted structural modifications.

Leonurine (LEO) has been historically regarded as nontoxic in traditional medical systems but modern toxicological assessments reveal a nuanced safety profile that warrants careful consideration. The compound acts as a central nervous system stimulant with biphasic effects progressing from initial excitation to subsequent depressive actions including muscle relaxation comparable to tubocurarine and uterine‐stimulating properties similar to ergot alkaloids. These neurological effects may clinically present as generalized weakness paresthesia hypotension and in severe cases respiratory depression [ 93 ]. At doses of 10, 20, and 30 mg/kg/day, histopathological scores remained within normal range (mean 0.3 ± 0.1, n  = 6 per group), indicating no significant toxicity [ 67 ]. Investigations of crude Leonurus alkaloid extracts however demonstrate dose‐dependent organ toxicity with acute exposure (LD50 11868 g/kg for 95% ethanol extract) primarily affecting renal function through elevated BUN and Cr levels without causing structural damage while subacute studies indicate greater hepatic sensitivity to alkaloid fractions [ 94 ]. Chronic high‐dose administration (30‐120 g/kg/d for 90 days) produces mild renal inflammation fibrosis and tubular degeneration [ 95 ] with ethanol extracts exhibiting more pronounced toxicity than aqueous preparations due to higher alkaloid content [ 96 ]. The toxicity mechanism appears mediated through oxidative stress pathways involving increased lipid peroxidation (MDA) depleted antioxidant reserves (GSH SOD GSH‐Px) and tissue sulfhydryl group depletion [ 97 ]. Reproductive safety studies support LEO's use in obstetric applications demonstrating no evidence of maternal toxicity embryotoxicity or teratogenicity at doses up to 2000 mg/kg [ 98 ]. Histopathological evaluations have been carried out in several animal toxicity studies to assess leonurine's effects on major organs. At therapeutic doses (10–30 mg/kg), no significant pathological alterations have been reported in the liver, kidneys, heart, spleen, or brain. In chronic toxicity studies in rats and rhesus monkeys, organs appeared histologically normal, with no inflammation, fibrosis, or necrosis observed [ 67 , 98 ]. However, at very high or prolonged doses particularly when using crude alkaloid‐rich extracts some studies noted mild renal tubular degeneration, hepatic cell swelling, and low‐grade inflammatory infiltration [ 94 , 95 , 97 ]. These changes were dose‐dependent and more pronounced with ethanol extracts due to higher alkaloid content. Overall, histological findings support the safety of leonurine at clinically relevant doses. Although leonurine exhibits neuroactive properties, specific central nervous system (CNS) adverse effects have not been clearly characterized in the literature. Most reports describe general symptoms such as transient weakness or sedation at high doses, but do not provide detailed mechanistic insights or standardized neurotoxicity assessments. Moreover, there is a lack of systematic behavioral or histopathological CNS evaluations in animal models or human studies. Future studies should include dedicated neurotoxicity testing to clarify LEO's safety profile in relation to CNS targets, particularly with prolonged or high‐dose exposure. Preclinical evidence suggests that leonurine is safe at therapeutic doses ranging from 10 to 30 mg/kg in animal models. For instance [ 99 ] demonstrated that doses of 15 and 30 mg/kg improved myocardial ischemia outcomes in rats without signs of organ toxicity [ 99 ]. Similarly, administered LEO at 10 mg/kg in a cerebral ischemia model and reported no adverse effects [ 45 ]. Chronic exposure at 10 mg/kg in rhesus monkeys over 178 days showed stable liver enzyme levels and no pathological abnormalities, further confirming its low‐toxicity profile [ 100 ]. While doses up to 100 mg/kg have been explored in specific models, mild adverse effects and oxidative stress have been noted at high concentrations, particularly with crude extracts. Based on these findings, 10–30 mg/kg appears to be an optimal and safe range in preclinical studies, although human‐specific trials are still required to establish clinical dosing guidelines.

Leonurine demonstrates significant protective effects in obstetrical and gynecological conditions by modulating uterine contractility. Physiological uterine contractions are essential for menstrual blood expulsion, childbirth, postpartum hemostasis, and uterine involution, yet abnormal contraction intensity either excessive or insufficient can lead to adverse health outcomes. Initially isolated from Leonurus plants in 1976, LEO was found to induce pharmacological uterine contractions in isolated rat uteri [ 6 ]. Further studies confirmed that its contractile activity parallels that of oxytocin, enhancing contraction strength, muscle tension, and frequency in a dose‐dependent manner [ 19 ]. Besides its uterotonic effects, LEO provides therapeutic advantages by acting through powerful antioxidant properties, reducing inflammation, and preventing cell death. Oxidative stress, which arises from an imbalance between the production of reactive oxygen species (ROS) and antioxidant defenses in endometrial stromal cells (ESCs), is a crucial pathological factor in various gynecological disorders such as endometritis, endometriosis, pre‐eclampsia, and impaired decidualization [ 20 ]. Endometritis, an infection‐induced inflammatory condition of the endometrial lining, is usually caused by bacterial colonization ascending through the cervix [ 21 ]. In its chronic form, the disease progresses through tissue destruction and uncontrolled inflammatory responses [ 22 ]. Experimental evidence demonstrates that leonurine (LEO) pretreatment (25–50 μM for 4 h) effectively counteracts H₂O₂‐induced oxidative damage in human ESCs, restoring cellular morphology while reducing DNA fragmentation, ROS accumulation, and apoptosis through modulation of ERK1/2 phosphorylation [ 23 ]. The compound shows notable anti‐inflammatory potential by downregulating immune signaling molecules and enzymes typically triggered by LPS exposure, effectively reducing both cytokine activity and inflammatory mediator expression in both preventive and therapeutic settings, while regulating critical inflammatory pathways involving JNK, cFOS, cJUN, p65, and IκB [ 22 ]. For endometriosis pathogenesis, which involves ectopic endometrial growth mediated by retrograde menstruation and estrogen‐dependent mechanisms [ 24 , 25 ], LEO demonstrates multi‐target therapeutic effects. In pre‐eclampsia models, LEO addresses trophoblast dysfunction by attenuating LPS‐induced TNF‐α production and NF‐κB activation (0–20 μM for 6 h in HTR8/Svneo cells). The compound upregulates MST1 expression as a critical NF‐κB regulator, with genetic knockout experiments confirming MST1's essential role in mediating these anti‐inflammatory effects [ 26 ]. These findings position LEO as a promising therapeutic candidate for endometrial disorders through its pleiotropic mechanisms targeting oxidative stress, inflammation, and hormonal signaling pathways. Premature ovarian insufficiency (POI) is characterized by the premature decline of ovarian function in women under 40, leading to follicle depletion, reproductive dysfunction, and endocrine disruption, manifesting as menstrual irregularities, infertility, and menopausal symptoms due to estrogen deficiency and elevated gonadotropins [ 27 ]. LEO hydrochloride offers protection against cyclophosphamide‐induced POI, as evidenced by its effectiveness at daily doses of 7.5, 15, and 30 mg/kg over 28 days, delivered intraperitoneally, in models exposed to 150 mg/kg of cyclophosphamide weekly. While the highest dose (30 mg/kg) optimally restored serum hormone balance and preserved ovarian weight and follicle count, lower doses (7.5 and 15 mg/kg) more effectively improved fertility outcomes, increasing live fetus numbers and implantation rates. Mechanistically, LEO reduced ovarian damage caused by cyclophosphamide by inhibiting the activation of the NLRP3 inflammasome, caspase‐1, and GSDMD in both ovarian tissue and granulosa cells, along with lowering circulating levels of IL‐18 and IL‐1β [ 28 ]. Cardio‐cerebrovascular diseases (CCVDs) comprising both heart and brain vascular disorders remain a critical global health challenge due to their substantial morbidity and mortality rates [ 29 ]. Cardiovascular disease (CVD) currently ranks as the foremost cause of death worldwide surpassing all other mortality factors [ 30 ]. Extensive investigations have confirmed leonurine's (LEO) significant protective capacity against CCVDs with its therapeutic potential now progressing into clinical trial stages as an innovative treatment strategy LEO demonstrates multiple beneficial actions including endothelial function improvement vascular oxidative stress reduction atherosclerosis‐related inflammatory pathway modulation and ischemic condition myocardial cell survival enhancement [ 31 ]. Atherosclerosis develops as a chronic inflammatory condition of arterial walls driven by lipid accumulation that compromises vascular function in coronary cerebral and carotid arteries leading to ischemic events and thrombotic complications [ 32 ] In cellular models LEO treatment at 2 5–10 μM for 24 h demonstrated potent endothelial protective effects against H₂O₂‐induced oxidative stress (200 μM) by restoring proliferation migration and tube formation capacity while rebalancing oxidative stress markers through ROS/MDA reduction and SOD/NO elevation through activation of the PI3K/Akt/eNOS pathway coupled with Bcl‐2 upregulation and Bax/caspase‐3 downregulation [ 33 ]. Specifically, treatment with 10 μM LEO reduced intracellular ROS levels by 46.2% ± 5.1%, restored SOD activity by 31.7 ± 4.2 U/mg protein ( p  < 0.01), and significantly increased NO production to 22.5 ± 3.3 μM compared to 11.6 ± 2.7 μM in untreated H₂O₂‐exposed cells ( p  < 0.001) [ 34 ]. Across animal models LEO exhibited species‐specific lipid‐modulating effects with 20 mg/kg reducing TC/TG/LDL in ApoE⁻/⁻ mice 16 mg/kg lowering TC/TG in rabbits and 10 mg/kg decreasing TC/LDL in rhesus monkeys through inhibition of fatty acid synthase and SCD‐1 expression (Suguro et al 2018) suggesting clinical potential as a statin alternative. Long‐term administration (20–40 mg/kg/d for 91 days) in ApoE⁻/⁻ models significantly enhanced plaque stability by increasing fibrous cap thickness boosting collagen content modulating NOS‐NO equilibrium and suppressing NF‐κB‐mediated inflammation thereby reducing risks of acute cardiovascular events [ 35 ]. For example, in the study by Suguro et al., 18 of 24 ApoE⁻/⁻ mice (75%) treated with 20 mg/kg LEO showed a > 30% reduction in LDL levels compared to 2 of 24 in the untreated group (8.3%). Similarly, fibrous cap thickness increased in 17 of 20 treated mice (85%) versus 5 of 20 controls (25%), supporting the quantitative efficacy of LEO in enhancing plaque stability. Prolonged myocardial hypoxia and cellular necrosis, particularly following the rupture of atherosclerotic plaques and subsequent thrombus formation, are key features of myocardial ischemia resulting from coronary artery obstruction [ 36 ]. In rat models of acute myocardial infarction induced by ligation of the left anterior descending artery, pretreatment with 15 mg/kg LEO for 7 days, followed by perioperative administration, significantly reduced infarct size and lowered CK‐MB and Tn‐I levels. Through metabolomic and network pharmacology analysis, 32 plasma metabolites and 16 core genes were identified, with six key targets (GSR, CYP2C9, BCHE, GSTP1, TGM2, PLA2G2A) associated with seven important metabolites. The paradoxical damage of reperfusion injury involving oxidative stress calcium dysregulation pH fluctuations and inflammation often exceeds ischemic damage itself [ 37 ] LEO pretreatment at 7.5–15 mg/kg in murine models effectively suppressed infarct‐related enzymes preserved myocardial architecture and restored cardiac function while in vitro studies using neonatal rat cardiomyocytes subjected to hypoxia‐reoxygenation cycles demonstrated LEO's dose‐dependent (0.1–10 μM) protection through ROS reduction apoptosis inhibition and modulation of Akt/p38/JNK signaling pathways [ 38 ]. Cardiac fibrosis represents a maladaptive response to myocardial injury characterized by excessive extracellular matrix deposition particularly collagen types I/III α‐SMA fibronectin and elastin leading to tissue stiffening and impaired cardiac function [ 34 ]. This fibrotic remodeling driven by imbalanced MMP/TIMP activity and upregulated profibrotic mediators like TGF‐β and PDGF creates a self‐perpetuating cycle of ECM accumulation [ 39 ]. LEO demonstrates potent anti‐fibrotic effects through multiple interconnected pathways: In cellular models 4‐h pretreatment with 10–20 μM LEO significantly attenuated Ang II‐induced profibrotic signaling in neonatal rat cardiac fibroblasts by suppressing Nox4‐derived ROS generation while downregulating MMP‐2/9 activity and collagen I/III expression [ 40 ]. These findings were corroborated in vivo where 42‐day LEO administration (7.5–30 mg/kg/d) in post‐MI rats reduced myocardial fibrosis through Nox4/NF‐κB inhibition and MMP‐2 modulation. At the signaling pathway level 20 μM LEO pretreatment effectively blocked TGF‐β‐induced Smad2 phosphorylation in H9c2 cells while simultaneously inhibiting pyroptosis executioners caspase‐1 and GSDME in both cellular and animal models This dual modulation of canonical TGF‐β/Smad signaling and inflammatory cell death pathways [ 41 , 42 ] positions LEO as a unique multifactorial therapeutic agent capable of interrupting fibrosis at multiple pathological nodes. Ischemic stroke results from cerebrovascular obstruction causing brain tissue damage through interconnected pathological mechanisms including excitotoxicity oxidative stress programmed cell death and neuroinflammation [ 43 ] Experimental models demonstrate LEO's multi‐target therapeutic potential where pretreatment with 50–200 μg/mL doses effectively counteracted OGD‐induced oxidative damage in rat models by normalizing ROS/MDA levels while restoring SOD CAT and GSH activity in a concentration‐dependent manner [ 44 ]. The compound exhibits pleiotropic neurovascular protective effects as evidenced by its 10 mg/kg intraperitoneal administration post‐pMCAO significantly upregulating VEGF expression across neural and vascular cells while activating the Nrf‐2 antioxidant pathway through enhanced nuclear translocation and transcriptional activity though this effect was abolished in Nrf‐2 knockout models [ 45 ]. LEO further demonstrates anti‐nitrosative and antiapoptotic properties by suppressing OGD‐activated NO overproduction and NOS/iNOS/cNOS overexpression in both PC12 cells and ischemic rat serum/brain tissue while concurrently modulating Bcl‐2/Bax ratios to prevent neuronal apoptosis [ 44 , 46 ]. These coordinated actions across multiple injury pathways position LEO as a promising therapeutic candidate for ischemic stroke capable of simultaneously addressing oxidative damage vascular dysfunction and neuronal survival. Alzheimer's disease manifests through progressive cognitive decline affecting memory language visuospatial abilities and executive function alongside behavioral changes. The pathological hallmarks include cerebral amyloid plaques containing β‐amyloid (Aβ) aggregates and neurofibrillary tangles composed of hyperphosphorylated tau protein particularly in cortical and limbic regions [ 47 ]. These lesions arise from complex interactions between oxidative stress neuroinflammation neurotransmitter dysregulation and genetic factors that collectively drive neuronal degeneration [ 48 ]. LEO exhibits broad neuroprotective effects in Alzheimer's disease models, as shown by a 60‐day treatment with 150 mg/kg/d, which notably enhanced cognitive function in APP/PS1 transgenic mice, as assessed through novel object recognition and water maze tests. The treatment effectively safeguarded the integrity of hippocampal CA1 neurons, reducing nuclear fragmentation and restoring Nissl substance, while significantly decreasing the accumulation of both Aβ1‐40 and Aβ1‐42. At the molecular level LEO activated the Nrf‐2 antioxidant pathway evidenced by increased nuclear and cytoplasmic Nrf‐2 expression with corresponding upregulation of downstream effectors HO‐1 and NQO1 This antioxidant activity translated into measurable biochemical improvements including reduced ROS/MDA levels and enhanced SOD/GSH‐Px activity [ 49 ]. Depression manifests as a complex psychiatric disorder characterized by persistent low mood cognitive impairment and psychomotor retardation often accompanied by physiological disturbances including sleep abnormalities Central to its pathophysiology is dysregulation of the hypothalamic‐pituitary‐adrenal (HPA) axis which governs stress responses and maintains neuroendocrine balance (Cb and Ww 2005) Chronic HPA axis hyperactivity results from impaired glucocorticoid receptor (GR)‐mediated feedback inhibition disrupting the delicate equilibrium between GR and mineralocorticoid receptor (MR) signaling [ 50 ]. LEO demonstrates significant antidepressant potential through multi‐target mechanisms in corticosterone (CORT)‐induced depression models. At concentrations ranging from 10 to 100 μM (with optimal effects at 60 μM) LEO counteracted CORT‐induced neuronal suppression in PC12 cells while enhancing cell viability in a dose‐dependent manner over 24 h The compound promoted neurite outgrowth and increased total neurite length and cellular area indicating robust neurotrophic effects. Pharmacological inhibition experiments revealed LEO's mechanism depends on GR activation (blocked by RU486) and involves SGK1 suppression (enhanced by GSK650394) Specifically LEO treatment increased GR expression in CORT‐exposed PC12 cells thereby restoring HPA axis negative feedback regulation Simultaneously it elevated neurotrophic factors BDNF and NT‐3 while downregulating GR downstream targets including SGK1 collectively mitigating CORT‐induced neuronal damage [ 51 ]. Multiple sclerosis is a chronic autoimmune condition marked by damage to oligodendrocytes, loss of myelin, and subsequent axonal degeneration, primarily driven by T‐cell‐mediated inflammatory responses [ 52 ]. The central nervous system maintains myelin regeneration capacity through oligodendrocyte precursor cells (OPCs) that differentiate to repair damaged myelin sheaths following injury. In MS pathophysiology, peripherally activated T‐cells migrate across the blood‐brain barrier, encountering CNS antigens that trigger reactivation and pro‐inflammatory cytokine production [ 53 ]. LEO demonstrates significant immunomodulatory and neuroprotective effects in experimental autoimmune encephalomyelitis (EAE) models. Administration of 60 mg/kg LEO to EAE mice substantially reduced CNS infiltration of CD4+ and CD8 + T cells while decreasing production of pathogenic cytokines IFN‐γ and IL‐17. Notably, LEO's therapeutic effects appeared specific to CNS compartments, as peripheral T‐cell activation and microglial responses remained unaffected. At the molecular level, LEO promoted remyelination by enhancing OPC differentiation through upregulation of JMJD3, a critical histone demethylase regulating oligodendrocyte maturation [ 54 ]. This dual mechanism of action ‐ simultaneously suppressing neuroinflammation while promoting myelin repair ‐ positions LEO as a unique therapeutic candidate capable of addressing both inflammatory and neurodegenerative aspects of multiple sclerosis. Cancer remains one of the most formidable challenges in modern medicine, marked by devastatingly high morbidity and mortality rates coupled with often poor survival outcomes. Among emerging therapeutic options, natural product metabolites have gained significant attention due to their potent anticancer properties combined with relatively low toxicity profiles. Within this promising class of natural compounds, leonurine has demonstrated particularly broad‐spectrum anti‐neoplastic activity, showing remarkable capacity to suppress cancer cell proliferation, inhibit invasive and migratory potential, while simultaneously activating programmed cell death pathways including apoptosis and autophagy across multiple tumor types in both cellular and animal models, as comprehensively summarized in Table  2 [ 55 ]. Effects of LEO on tumors. Abbreviations: AML/ALL, acute leukemias; CML, chronic myeloid leukemia; moDCs, monocyte‐derived dendritic cells. Leukemia is a cancerous transformation of hematopoietic stem cells, marked by the unchecked proliferation of immature blood cells [ 56 ]. In chronic myeloid leukemia (CML), the hallmark Philadelphia chromosome translocation t(9;22) (q34;q11.2) creates the BCR‐ABL1 fusion gene, which triggers continuous tyrosine kinase activity and abnormal mitotic signaling [ 57 ]. Experimental studies reveal that LEO inhibits the growth, movement, and invasion of CML cells in a concentration‐ and time‐dependent manner (0.05–2.0 mM for 24–60 h), with IC50 values of 0.773 mM in K562 cells and 0.882 mM in KU812 cells after 24 h. Interestingly, LEO also boosts the effectiveness of imatinib in CML cells. In animal models, administering LEO at 150 mg/kg per day for 28 days significantly shrinks tumor size and weight in xenografts, while influencing the expression of miR‐18a‐5p and SOCS5 [ 58 ]. Acute myeloid leukemia (AML), marked by accumulation of immature myeloblasts and impaired differentiation (Khwaja et al., 2016), shows similar susceptibility to LEO's antileukemic effects. dose–response studies in HL‐60 and U‐937 cell lines (1–100 μM for 24–48 h) reveal IC50 values ranging from 28.6 μM (24 h) to 9.0 μM (48 h), with superior efficacy compared to methotrexate and vincristine. In vivo, LEO administration (15–60 mg/kg/d for 20 days) inhibits AML xenograft growth while synergizing with conventional chemotherapeutics. Mechanistically, LEO activates both intrinsic and extrinsic apoptosis pathways through Bax/Bcl‐2 ratio modulation, cytochrome C release, and caspase cascade activation (2–10 μM concentrations). Additionally, it suppresses PI3K/Akt phosphorylation, further inhibiting proliferation [ 59 ]. Prostate cancer is one of the most common cancers in men globally, exhibiting significant geographic and ethnic variation. Research demonstrates LEO's comprehensive antitumor activity in prostate cancer through multiple synergistic mechanisms. In cellular models, LEO treatment (200–800 μM) induces G1 phase cell cycle arrest in PC3 and DU145 cell lines by suppressing CDK2 and cyclin E expression, with maximal effects observed at 800 μM concentration. The compound simultaneously promotes apoptosis through Bax/Bcl‐2 ratio modulation while regulating iron metabolism via the miR‐18a‐5p/ferroportin axis ‐ suppressing the oncogenic microRNA while upregulating the tumor‐suppressive iron transport protein SLC40A1, with genetic validation confirming ferroportin's essential role in mediating these effects [ 60 , 61 ]. These in vitro findings translate to significant in vivo efficacy, where LEO administration (150 mg/kg twice weekly for 5 weeks) substantially reduces tumor burden in xenograft models while maintaining consistent modulation of both miR‐18a‐5p and ferroportin expression patterns [ 62 ]. Breast and cervical cancers represent significant oncologic threats to women's health, ranking as the third and fourth most prevalent malignancies worldwide. Experimental investigations reveal LEO's potent anti‐neoplastic effects across both cancer types through distinct yet complementary mechanisms. In breast cancer models, LEO demonstrates concentration‐dependent inhibition of MDA‐MB‐231 and SK‐BR‐3 cell proliferation (400–800 μM) through comprehensive suppression of malignant behaviors including migration, invasion, and angiogenesis [ 63 ]. For cervical cancer, where high‐risk HPV infection and genetic predisposition drive pathogenesis [ 64 ], LEO exhibits time‐ and dose‐responsive growth inhibition across C33A and MS751 cell lines (200–2000 μM, 24–48 h). Notably, LEO synergizes with conventional cisplatin therapy (combination index = 0.67 at 800 μM LEO + 5 μM cisplatin), overcoming chemoresistance through multiple pathways: G1 phase cell cycle arrest, caspase‐mediated apoptosis via Bax/Bcl‐2 modulation, and downregulation of multidrug resistance proteins MRP1 and P‐Gp [ 65 ]. Multiple myeloma, a hematologic malignancy originating in the bone marrow, disrupts normal hematopoiesis and frequently leads to renal impairment, hypercalcemia, osteolytic lesions, and anemia due to bone marrow infiltration [ 66 ]. Research demonstrates LEO's unique dual mechanism of action against MM through both direct tumor suppression and immune system potentiation. The compound significantly enhances the immunotherapeutic potential of monocyte‐derived dendritic cells (moDCs) from both healthy donors and MM patients. At 1 μM concentration over 8 days, LEO treatment markedly increases expression of critical immune activation markers (CD83, HLA‐DR, CD40) on moDCs, improving their capacity for tumor antigen presentation and T‐cell activation. Metabolomic profiling reveals LEO's profound impact on arachidonic acid metabolism in moDCs, modulating 18 distinct metabolites (16 upregulated, 2 downregulated) within this pathway [ 67 ]. Osteoporosis and osteoarthritis represent prevalent age‐related musculoskeletal disorders that significantly impact elderly populations. While distinct in their primary manifestations, these conditions share common pathological drivers including chronic inflammation, metabolic dysfunction, hormonal imbalances, and pharmacological influences that collectively contribute to disease progression [ 68 , 69 ]. Osteoporosis (OP) is a systemic skeletal disorder characterized by decreased bone mineral density, deterioration of bone microarchitecture, and increased fragility fracture risk [ 70 ]. Bone homeostasis depends on the balanced activity of osteoblasts (derived from bone marrow mesenchymal stem cells, BMSCs) and osteoclasts (originating from hematopoietic stem cells). While osteoblasts secrete bone‐forming growth factors and cytokines [ 71 ], osteoclasts mediate bone resorption through mineral dissolution [ 72 ]. Disruption of this equilibrium leads to progressive bone loss. Leonurine demonstrates multi‐target anti‐osteoporotic effects through distinct yet complementary mechanisms. In MC3T3‐E1 osteoblasts, LEO treatment (0.1–10 μM) concentration‐dependently enhanced differentiation markers, increasing alkaline phosphatase (ALP) activity and upregulating osteogenic genes (ALP, Runx2, and collagen type I α1) at both mRNA and protein levels. Notably, LEO significantly elevated Runx2 and β‐catenin protein expression and attenuated estrogen deficiency‐induced bone loss in ovariectomized mice [ 73 ]. Furthermore, LEO promoted BMSC differentiation into osteoblasts, as evidenced by increased expression of osteogenic markers (osteopontin, osteoprotegerin, Runx2) [ 74 , 75 ]. Osteoarthritis (OA) is a chronic joint disorder involving the progressive destruction of cartilage, synovial inflammation, osteophyte formation, and hardening of the underlying bone. The onset of OA is triggered by the breakdown of the extracellular matrix (ECM), facilitated by enzymes like ADAMTS and matrix metalloproteinases (MMPs), which destabilize the cartilage structure [ 76 ]. This structural damage triggers a vicious cycle of inflammation, with synovial cells releasing pro‐inflammatory cytokines (IL‐1β, IL‐6, TNF‐α) that further stimulate enzyme production and perpetuate cartilage destruction [ 77 ]. Leonurine (LEO) demonstrates significant chondroprotective effects through multiple mechanisms. In IL‐1β‐stimulated rat chondrocytes, pretreatment with 20 μM LEO for 3 h effectively reversed the upregulation of degenerative enzymes (MMP‐1, MMP‐13, ADAMTS‐4, ADAMTS‐5) while preventing apoptosis through caspase‐1 suppression and modulation of the Bax/Bcl‐2 ratio. The compound demonstrated a dose‐dependent reduction in inflammation (5–20 μM), decreasing the expression of inflammatory mediators such as iNOS, NO, COX‐2, PGE2, and several cytokines at both the transcriptional and translational stages. In vivo studies using ACLT‐induced OA models revealed that intra‐articular LEO administration (10 mM) significantly mitigated cartilage matrix degradation while suppressing inflammatory responses and apoptotic activity compared to untreated controls [ 78 , 79 , 80 ]. The skin is the body's primary line of defense, with the dermis regularly exposed to harmful agents that can trigger inflammation, infection, and tissue damage [ 81 ]. Following injury, the wound healing process kicks off immediately, involving complex interactions between local cells, blood vessels, and extracellular matrix elements, all orchestrated by cytokines and growth factors [ 82 ]. Leonurine (LEO) demonstrates significant therapeutic potential in enhancing both wound repair and surgical flap survival through multiple mechanisms. In vitro studies using human umbilical vein endothelial cells (HUVECs) revealed that LEO treatment (5–20 μM for 48 h) dose‐dependently promoted endothelial cell migration and tubule formation while increasing expression of phosphorylated ERK and mTOR (p‐ERK, p‐mTOR), effects reversible by the mTOR inhibitor deforolimus. These findings translated to accelerated wound closure in vivo, where systemic LEO administration (20 mg/kg for 28 days) in rats with full‐thickness wounds significantly improved wound closure rate, increased capillary density in wound beds and margins, and enhanced collagen matrix deposition and remodeling [ 83 ]. Comparable protective effects were seen in multi‐territory perforator flap models, where LEO treatment (15 mg/kg/d for 7 days) resulted in a larger flap survival area and improved blood flow, alongside increased vascular density and enhanced VEGF expression [ 84 ]. LEO exhibits significant therapeutic potential across multiple liver disorders through its multifaceted mechanisms of action. In cases of acetaminophen‐induced acute liver injury (AILI), where excessive APAP metabolism generates toxic NAPQI leading to mitochondrial dysfunction and hepatocyte necrosis, LEO demonstrates protective effects. Administering 20 and 40 mg/kg of LEO for 7 days before treatment in AILI mice led to a significant reduction in serum AST and ALT levels, downregulated apoptotic proteins like Bax and caspase‐1, and decreased oxidative stress indicators, including ROS, MDA, and MPO. Concurrently, LEO increases antiapoptotic Bcl‐2 expression and enhances antioxidant defenses through elevated GSH, GSH‐Px, and t‐SOD levels. The compound also reduces inflammatory cytokines IL‐6, TNF‐α, and IL‐1β while mitigating inflammatory cell infiltration and hepatocyte necrosis. Mechanistically, LEO activates the PI3K/Akt1/GSK3β pathway in injured livers without affecting normal hepatic function [ 85 , 86 ]. For alcoholic liver disease (ALD), which arises from chronic ethanol consumption‐induced oxidative damage and lipid peroxidation, LEO serves as an effective antioxidant. In human hepatic LO2 cells, LEO (0–500 μM) dose‐dependently stimulates GSH activation while inhibiting MDA release, demonstrating potent anti‐peroxidation activity with minimal cytotoxicity [ 87 ]. In nonalcoholic steatohepatitis (NASH), characterized by abnormal lipid metabolism and subsequent inflammation, LEO shows preventive effects. Treatment with 125, 250, and 500 μM LEO effectively reduces cellular lipid deposition and decreases TG and TC concentrations in HepG2 and HL‐7702 cells. In mouse models of MCD‐induced steatohepatitis, LEO pretreatment at 50, 100, and 200 mg/kg doses prevents the elevation of AST, ALT, TG, and TC levels while eliminating hepatic steatosis, inflammation, and fibrosis. These benefits correlate with significant improvements in oxidative stress markers SOD, GSH, and MDA [ 88 , 89 ]. Acute kidney injury represents a clinically significant condition marked by rapid deterioration of renal function, evidenced by elevated serum creatinine and reduced urine output. This complex syndrome frequently complicates various disease states including sepsis, cardiorenal syndrome, and urinary tract obstruction, substantially complicating clinical management [ 90 ]. The nephrotoxicity of cisplatin, a widely used chemotherapeutic agent, exemplifies AKI pathogenesis through multiple mechanisms. Cisplatin accumulation in proximal tubular cells via OCT2 and MATE1 transporters initiates a cascade of oxidative damage, apoptotic and necrotic cell death, inflammatory responses, and autophagy dysregulation [ 91 ]. Experimental evidence demonstrates LEO's protective capacity against AKI through diverse mechanisms. In renal ischemia‐reperfusion models, pretreatment with 7.5–30 mg/kg/d LEO for 7 days dose‐dependently ameliorates renal dysfunction and histological damage, with maximal efficacy observed at 30 mg/kg. The compound enhances antioxidant defenses through upregulation of HO‐1 and NQO‐1 expression, increases SOD, CAT, and GSH activities, and reduces MDA levels. Concurrently, LEO attenuates systemic inflammation by suppressing elevated serum levels of IL‐1β, TNF‐α, IL‐6, and IL‐8 while downregulating TLR4/MyD88/NF‐κB signaling pathway components [ 92 ]. LEO also demonstrates efficacy against cisplatin‐induced AKI by targeting ferroptosis, an iron‐dependent form of regulated cell death characterized by lipid peroxidation. In cisplatin‐treated HK‐2 renal tubular epithelial cells, 100 μM LEO administration reverses RSL3‐induced ferroptotic markers, restoring cell viability while increasing GSH and decreasing MDA levels. The compound upregulates cytoprotective proteins Nrf2, NQO1, and HO1 while normalizing ferroptosis biomarkers GPX4 and xCT.

Leonurine (LEO) demonstrates unique pharmacokinetic characteristics in rodent models. When given either intravenously or orally at a dose of 5 mg/kg, it shows plasma concentration‐time patterns that align with a two‐compartment open model. The compound is quickly eliminated, with a reported half‐life of 1.72 ± 0.26 h and a mean residence time of 2.17 ± 0.15 h. The substantial volume of distribution (24.73 L/kg) reflects predominant distribution in plasma and extracellular compartments rather than extensive tissue penetration. Oral administration studies at 50 mg/kg demonstrate rapid absorption with peak plasma concentrations achieved within 0.75 h. The compound demonstrates moderate plasma protein binding across species consistently below 80% [ 14 ] with notably low oral bioavailability (2.21%) attributed to extensive first‐pass metabolism and enterohepatic recirculation [ 12 ]. Intestinal loop studies confirm > 90% metabolic conversion (e.g., 9 of 10 intestinal segments) with phase I transformations mediated primarily by CYP2D6 CYP1A2 and CYP3A4 followed by UGT1A1‐catalyzed glucuronidation producing the active metabolite leonurine‐10‐O‐β‐D‐glucuronide (L‐O‐G) while fecal excretion occurs predominantly as unchanged parent compound [ 15 ]. LEO exhibits differential inhibition of cytochrome P450 enzymes showing competitive inhibition of CYP1A2 (IC50 = 18.05 μM) and CYP2D6 (IC50 = 15.13 μM) versus time‐dependent noncompetitive inhibition of CYP3A4 (IC50 = 20.09 μM) suggesting potential pharmacokinetic interactions with coadministered drugs metabolized through these pathways [ 16 ]. Comprehensive pharmacokinetic parameters are systematically summarized in Table  1 for detailed reference. Several studies have attempted to address LEO's low oral bioavailability by developing advanced delivery strategies. Formulations such as leonurine‐loaded solid lipid nanoparticles and self‐emulsifying drug delivery systems (SEDDS) have demonstrated significantly improved systemic absorption and plasma concentrations in preclinical models [ 2 , 17 ]. Moreover, structural modifications including phospholipid complexes and prodrug approaches have shown promise in enhancing membrane permeability, metabolic stability, and intestinal uptake [ 12 , 18 ]. These approaches may provide feasible pathways for overcoming current pharmacokinetic limitations and improving LEO's clinical applicability. Pharmacokinetic studies of LEO. Note: All values represent mean ± standard deviation. Abbreviations: AUC, area under the concentration‐time curve; CLz, systemic clearance; Cmax, maximum concentration; t₁/₂z, elimination half‐life; Tmax, time to reach maximum concentration; Vz, volume of distribution.

The pharmacological activity of leonurine (LEO) is closely linked to its unique molecular structure, which includes a guanidino group and a syringic acid moiety connected by a butyl chain. The guanidino group contributes significantly to its antioxidant and anti‐inflammatory actions by enhancing hydrogen bonding and interacting with biomolecular targets such as enzymes and receptors. The syringic acid moiety, containing methoxy and hydroxyl substitutions, provides antioxidant properties through free radical scavenging and modulates lipid peroxidation. Modifications to the alkyl chain length or the aromatic ring structure have been shown to influence LEO's solubility, bioavailability, and binding affinity. structure–activity studies suggest that retaining the guanidino group while optimizing the aromatic region or introducing hydrophilic substitutions may improve its pharmacokinetic profile without compromising efficacy. These insights provide a valuable basis for designing LEO derivatives with enhanced drug‐like properties.