Glycyrrhizin (Glycyrrhizic Acid)—Pharmacological Applications and Associated Molecular Mechanisms

Abstract

Background/Objectives: Natural products, especially plant metabolites, play a crucial role in drug development and are widely used in medicine, cosmetics, and nutrition. The present review aims to provide a comprehensive overview of the pharmacological profile of Glycyrrhizin (GL), with a specific focus on its molecular targets. Methods: Scientific literature was thoroughly retrieved from reputable databases, including Scopus, Web of Science, and PubMed, up to 30 July 2025. The keywords “glycyrrhizin” and “glycyrrhizic acid” were used to identify relevant references, with a focus on pharmacological applications. Studies on synthetic analogs, non-English publications, non-pharmacological applications, and GL containing crude extracts were largely excluded. Results: Glycyrrhizin, the major bioactive constituent of Glycyrrhiza glabra, exhibits diverse pharmacological activities, including anti-inflammatory, antiviral, hepatoprotective, antitumor, neuroprotective, and immunomodulatory effects. These actions are primarily mediated through the inhibition of high-mobility group box 1 (HMGB1) and the modulation of key signaling pathways, including nuclear factor kappa B (NF-κB), mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase/protein kinase B (PI3K/Akt), and various cytokine networks. As a result of its therapeutic potential, GL-based formulations, including Stronger Neo-Minophagen C, and GL-rich extracts of G. glabra are commercially available as pharmaceutical preparations and food additives. Conclusions: Despite its therapeutic potential, the clinical application of GL is limited by poor oral bioavailability, metabolic variability, and adverse effects such as pseudoaldosteronism. Hence, careful consideration of pharmacokinetics and safety is essential for translating its therapeutic potential into clinical practice.

1. Introduction

Glycyrrhizin (GL), also known as glycyrrhizic acid, is a naturally occurring oleanane-type triterpenoid saponin predominantly found in the roots of licorice (Glycyrrhiza glabra), where it constitutes approximately 10% of the plant’s dry weight [1]. In addition to G. glabra, other species such as G. uralensis and G. inflata also serve as sources of GL, albeit in comparatively lower concentrations [2]. GL, chemically identified as (3β,20β)-20-carboxy-11-oxo-30-norolean-12-en-3-yl 2-O-β-D-glucopyranuronosyl-α-D- glucopyranosiduronic acid, consists of a triterpenoid aglycone, glycyrrhetinic acid, linked to a disaccharide composed of glucuronic acid units (Figure 1). Well-known for its intensely sweet taste, estimated to be 50 to 100 times sweeter than sucrose, it has a slow onset of sweetness with a prolonged aftertaste. Beyond its use as a natural sweetener and flavoring agent, GL exhibits a wide range of therapeutic properties, including antiviral, anti-inflammatory, antitumor, antimicrobial, and hepatoprotective effects [2].

Glycyrrhiza glabra L. (licorice) belongs to the family Leguminosae and is native to southern Europe and parts of Asia. It is one of the most widely used and historically revered medicinal plants. Known for its sweet flavor, licorice has served both culinary and medicinal purposes since ancient times [3]. Civilizations such as the Egyptians, Romans, and Greeks consumed licorice-infused beverages for their thirst-quenching properties. In traditional systems like Ayurveda (where it is known as Mulethi) and traditional Chinese medicine (as Gancao), it has long been valued for managing respiratory conditions such as asthma, bronchitis, and dry cough, as well as for its general anti-inflammatory and detoxifying properties [4]. In addition to GL, licorice contains other bioactive constituents, such as chalcones, flavonoids, and saponins, that exhibit diverse pharmacological activities, including anti-inflammatory, antioxidant, antitumor, hepatoprotective, and immunomodulatory effects [5]. Modern applications range from pharmaceutical formulations to food additives, herbal teas, and tobacco products [6].

Physically, GL appears as a tan to white powder depending on its purity. It has a molecular weight of 822.94 g/mol and a molecular formula of C₄₂H₆₂O₁₆. It is an amphiphilic molecule with a melting point of approximately 220 °C, comprising a hydrophilic component (glucuronic acid residues) and a hydrophobic component (glycyrrhetinic acid). This amphiphilic nature enables it to influence surface tension and form micellar structures. GL is sparingly soluble in water (1–10 mg/mL at 20 °C), although its solubility increases significantly in the form of salts such as potassium, magnesium, and calcium glycyrrhizinates. It remains relatively stable within a pH range of 4–9 but degrades under prolonged heating or in highly acidic conditions. Chemically, it behaves as a weak acid, with three pKa values (2.7, 2.8, and 4.7), and exists in two stereoisomeric forms (18α and 18β) which differ in hydrogen orientation at C-18 [7,8]. Although 18α-glycyrrhizin is thermodynamically more stable, especially under aqueous alkaline conditions where 18β can epimerize to 18α, the 18β isomer is the predominant natural form and exhibits greater pharmacological activity. The α-form is a minor licorice root component, eliminated faster from the body, and often less potent. Interestingly, molecular modeling shows that 18α isomer forms more stable complexes with proteins like HMGB1, but this does not necessarily translate to higher biological activity [9].

The diverse pharmacological activities of GL are closely linked to its structural features. The 18β-glycyrrhetinic acid (18β-GA) moiety, its principal metabolite, plays a key role in mediating its bioactivity. Structural modifications at key positions, particularly the C3 hydroxyl and C30 carboxyl groups, significantly affect its molecular interactions and, consequently, its therapeutic potential. Additionally, alterations such as the modification of the C11–C12 double bond have been shown to enhance or reduce specific pharmacological effects [10,11].

2. Pharmacological Applications of Glycyrrhizin

GL has demonstrated a broad spectrum of pharmacological activities, supported by both preclinical and clinical studies. It exhibits potent anti-inflammatory properties by modulating key inflammatory mediators such as tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6, and the nuclear factor kappa B (NF-κB) signaling pathway [12]. Its hepatoprotective effects have been extensively documented in models of liver injury, where GL reduces inflammation, oxidative stress, and fibrosis, often through inhibition of CD4⁺ T cell proliferation and enhancement of IL-10 and interferon-gamma (IFN-γ) expression [13]. GL also displays notable antiviral properties, acting against viruses such as hepatitis C virus (HCV), herpes simplex virus (HSV), and SARS-CoV2 by inhibiting viral replication and modulating host immune responses [14]. Furthermore, its anticancer potential across various cancer types is attributed to the induction of apoptosis, inhibition of cell proliferation, and activation of peroxisome proliferator-activated receptor gamma (PPARγ) [15]. In addition, GL possesses antioxidant, neuroprotective, anti-diabetic, and cardioprotective properties in various in vitro and in vivo models. The detailed pharmacological activities of GL are discussed below.

2.1. Effect on Different Inflammatory Conditions

Glycyrrhizin (GL) exhibits notable anti-inflammatory and analgesic properties through multiple mechanisms, including inhibition of HMGB1/2 phosphorylation, modulation of nitric oxide (NO) and prostaglandin E2 (PGE2) production, and regulation of key pro-inflammatory cytokines such as TNF-α, IL-6, iNOS, and COX-2 (Figure 2). At the molecular level, GL binds directly to HMGB1/2, inhibiting their phosphorylation by CK-I and PKC, reducing DNA-binding capacity, and thereby suppressing inflammation [16]. In macrophages (RAW 264.7 cells), GL exerts a concentration-dependent effect on NO and PGE2, stimulating their production at low concentrations and inhibiting it at higher concentrations, while also scavenging superoxide anions without affecting DPPH or NO radicals [17].

In vivo, GL alleviates inflammation in various models, including xylene-induced ear edema, carrageenan-induced paw edema, acetic acid-induced vascular permeability, and nociception in the acetic acid and formalin tests, though it shows no effect in the hot plate test. It also inhibits phospholipase A2 (PLA2) activity and platelet aggregation [18,19], and reduces inflammatory mediators such as CC-chemokine ligand 2 (CCL2) in systemic inflammatory response syndrome (SIRS), while suppressing cytokine release from splenic T cells [20]. Topically, GL demonstrates dose-dependent inhibition of granuloma formation in the cotton pellet granuloma pouch test, comparable to indomethacin [21].

GL also modulates inflammasome pathways, particularly NLRP3, via HMGB1 and NF-κB, making it as a clinically used, non-specific NLRP3 inhibitor. This provides a safer and well-tolerated alternative to potent experimental inhibitors like MCC950, which are limited by hepatotoxicity, and broadly acting agents like minocycline, which show mixed clinical efficacy and lack specificity. These multifaceted mechanisms showed its therapeutic potential in managing acute and chronic inflammatory conditions.

The following sections outline the various inflammatory conditions in which GL has shown potential, along with the underlying mechanisms of its activity.

2.1.1. Effect on HMGB1 Secretion and Its Cytokine Activities

HMGB1 protein is a key pro-inflammatory mediator implicated in various pathological conditions, including sepsis, arthritis, endotoxemia, and autoimmune disorders. GL has been shown to strongly inhibit the pro-inflammatory activities of HMGB1. Specifically, it suppresses HMGB1’s chemoattractant and mitogenic effects, thereby reducing immune cell recruitment and activation. It also modestly inhibits HMGB1’s DNA-binding activity, which contributes to its pro-inflammatory functions. Biophysical studies have revealed that GL binds directly to HMGB1, with a dissociation constant of approximately 150 μM. The compound interacts with the concave surfaces of HMGB1’s HMG boxes, suggesting a structural basis for its anti-inflammatory effects and providing a rationale for the design of more potent HMGB1-binding derivatives [22]. Furthermore, GL inhibits HMGB1-induced monocyte migration and promotes apoptosis of activated immune cells, thereby suppressing the expression of inflammatory mediators such as MCP-1 and Mcl-1. These effects are primarily mediated through inhibition of the NF-κB and MAPK/ERK signaling pathways [23].

In animal models of endotoxemia, GL has been shown to mitigate HMGB1-driven organ injury and improve systemic hemodynamics. It effectively reduces circulating HMGB1 levels and downregulates the production of pro-inflammatory cytokines such as TNF-α and IL-6. The underlying mechanism involves suppression of the NF-κB and MAPK pathways, both of which are critical in mediating the inflammatory response during endotoxemia [24,25]. Supporting evidence from in vitro studies further highlights GL’s role as a direct modulator of HMGB1. In LPS-stimulated RAW 264.7 macrophage cells, GL inhibited HMGB1 secretion and prevented its translocation from the nucleus to the cytoplasm, an essential step in initiating the inflammatory cascade. Additionally, GL reduced NF-κB activation and subsequent release of inflammatory cytokines, reinforcing its therapeutic potential in endotoxemia [26,27]. In a model of autoimmune thyroiditis, Li et al. [28] demonstrated that GL significantly reduced thyroid inflammation in NOD.H-2h4 mice. Following induction of thyroiditis via 0.005% sodium iodide, mice were treated with GL (20 mg/kg/day) for 4 weeks. This intervention markedly reduced the expression of HMGB1, TNF-α, IL-6, and IL-1β, and alleviated lymphocytic infiltration in the thyroid gland. GL also downregulated key inflammatory markers such as TLR2, MyD88, and NF-κB, suggesting that its protective effect in autoimmune thyroiditis is mediated via inhibition of the TLR2-HMGB1 signaling axis.

2.1.2. Effect on Prostaglandin E2 Production

GL inhibits the production of PGE2 in activated rat peritoneal macrophages. Preincubation with GL enhances its inhibitory effect. At doses up to 3 mg/mL, GL effectively reduces PGE2 production without causing cytotoxicity. In contrast, glycyrrhetinic acid (the aglycone) at 100 μg/mL inhibited PGE2 production but caused significant cell detachment, indicating a toxic effect at higher concentrations [29].

2.1.3. Effect on Neutrophil Functions and ROS Generation

GL decreases ROS generation by neutrophils, including superoxide anions (O₂⁻), hydrogen peroxide (H₂O₂), and hydroxyl radicals (OH^•), in a dose-dependent manner. However, GL does not scavenge ROS in a cell-free system (e.g., xanthine-xanthine oxidase), nor does it affect neutrophil chemotaxis or phagocytosis. These results suggest that GL modulates neutrophil function by inhibiting ROS production rather than acting as a direct ROS scavenger [30].

2.1.4. Effect on Neutrophil-Induced Generation of Alternatively Activated Macrophages

In patients with severe burn injuries, where immune suppression and the generation of alternatively activated macrophages (M2Mφ) is common, GL inhibits M2Mφ generation. When PMN-II (immunosuppressive neutrophils) were cultured with resident macrophages (R-Mφ) in the presence of GL, M2Mφ were not generated. GL also suppressed the production of interleukin-10 and CCL2 by PMN-II, which are key effector molecules in M2Mφ generation [31].

2.1.5. Effect on Inflammatory Responses Mediated by Toll-like Receptors

GL attenuates pro-inflammatory responses induced by Toll-like receptors (TLR), including TLR3, TLR4, and TLR9. In RAW 264.7 macrophages, GL inhibited the induction of pro-inflammatory mediators caused by TLR9 agonists (CpG-DNA) and other TLR ligands. The mechanism involves blocking the NF-κB pathway and the MAPK signaling cascade. GL impairs the internalization of TLR4 and the cellular uptake of CpG-DNA. Its anti-inflammatory action was found to be mediated by interfering with membrane-dependent receptor signaling [32].

2.1.6. Effect on Salmonella enterica Serovar Typhimurium-Induced Injury

GL shows protective effects against Salmonella-induced inflammation in mice. GL alleviated weight loss and intestinal mucosal injury, reduced Salmonella enterica Serovar Typhimurium (ST) colonization in the ileum, colon, and translocation to the liver and spleen. Although GL did not inhibit bacterial growth directly, it significantly decreased pro-inflammatory cytokine secretion (IFN-γ, TNF-α, IL-6) and increased anti-inflammatory IL-10 secretion. GL also modulated gut microbiota, enriching beneficial species like Parabacteroides and Anaerotruncus. These findings suggest GL enhances immune function and modulates intestinal flora to protect against ST infection [33].

2.1.7. Effect on Traumatic Spinal Cord Injury

GL has demonstrated neuroprotective effects in various neurological conditions, particularly in models of traumatic spinal cord injury (SCI). Several studies have shown that GL confers therapeutic benefits by inhibiting NLRP3 inflammasome activation, reducing microglial M1 polarization, and promoting M2 polarization. This immune modulation contributes to attenuated neuroinflammation and enhanced functional recovery in SCI models [34]. For instance, oral administration of GL (10 mg/kg) in male Sprague-Dawley rats following SCI significantly improved behavioral outcomes. This was accompanied by a marked reduction in pro-inflammatory markers such as NLRP3, IL-1β, and IL-18. Concurrently, GL increased the expression of M2 microglial polarization markers (CD206, Arg-1) while decreasing M1 markers (CD86, iNOS) in the injured spinal cord. These findings indicate that GL’s regulation of neuroinflammatory pathways may serve as a potential therapeutic strategy for SCI [34].

In another study, using a mouse model of SCI induced by vascular clips at the T5–T8 level (laminectomy), intraperitoneal administration of GL (10 mg/kg) significantly reduced secondary inflammation and tissue damage. This was reflected by decreased levels of oxidative stress markers such as nitrotyrosine, iNOS, and poly(ADP-ribose). Moreover, GL treatment attenuated apoptosis—as evidenced by favorable shifts in Bax and Bcl-2 expression—and resulted in improved motor recovery scores. These results further support GL’s anti-inflammatory and anti-apoptotic effects, reinforcing its promise as a therapeutic agent for SCI [35].

2.1.8. Effect on Ischemia/Reperfusion Injury

GL has demonstrated significant protective effects across various ischemia/reperfusion (I/R) injury models, including those involving the spinal cord, skeletal muscle, and kidneys. Its anti-inflammatory and cytoprotective properties are central to its therapeutic potential in mitigating tissue damage caused by I/R. In a rabbit model of limb I/R injury, GL administration (both before and after ischemia, intravenously) effectively reduced tissue edema and necrosis. Treatment significantly limited the increase in limb circumference and decreased the number of necrotic muscle fibers observed in histological analyses. GL also lowered myeloperoxidase (MPO) activity, a marker of neutrophil infiltration, and suppressed the expression of P- and E- selectins, which mediate neutrophil adhesion. These findings suggest that GL can help prevent peripheral I/R injury through anti-inflammatory and anti-edematous mechanisms [36].

In spinal cord I/R injury models, GL (10 mg/kg) markedly reduced neuroinflammation, apoptosis, and locomotor deficits in C57BL/6 mice. These effects were attributed to inhibition of NF-κB activation, downregulation of pro-inflammatory cytokines such as TNF-α and IL-1β, and reduced neutrophil infiltration into the spinal cord. A key mechanism involved GL’s inhibition of HMGB1, a critical mediator of post-ischemic inflammation, thereby preserving neural tissue and enhancing functional recovery. Similarly, in skeletal muscle I/R injury, GL conferred protection by attenuating oxidative and inflammatory damage. In a rabbit model, treatment with GL significantly decreased serum levels of injury biomarkers, including creatine kinase, lactate dehydrogenase (LDH), and MPO. Histopathological assessments confirmed improved muscle integrity, with fewer necrotic fibers, supporting its efficacy in reducing muscle damage via antioxidant and anti-inflammatory pathways [37,38].

2.1.9. Effect on Subarachnoid Hemorrhage and Inflammatory Brain Injury

GL has shown promise as an inhibitor of HMGB1, a key mediator in the inflammatory response after subarachnoid hemorrhage (SAH). In a rat SAH model, GL administration (15 mg/kg) significantly improved neurological scores, reduced HMGB1 expression, and inhibited blood–brain barrier permeability. Furthermore, GL reduced neuronal apoptosis and the levels of inflammatory cytokines (TNF-α, IL-1β), and hence, make it a potential candidate for the treatment of SAH-related brain injury [39].

2.1.10. Effect on Lipopolysaccharide-Induced Inflammation

GL has demonstrated potent anti-inflammatory effects in response to lipopolysaccharide (LPS), a key endotoxin involved in systemic inflammation and sepsis. In LPS-stimulated RAW 264.7 macrophages, GL significantly reduced the production of pro-inflammatory cytokines, including IL-6 and TNF-α. This immunosuppressive action was mediated by the inhibition of the TLR4/MD-2 complex formation, which in turn blocked the downstream NF-κB signaling pathway. Additionally, GL attenuated the activation of mitogen-activated protein kinases (MAPKs), including JNK, p38, and ERK, further suppressing the inflammatory cascade [40]. In a complementary in vitro model of subacute ruminal acidosis induced by LPS in goat ruminal epithelial cells, GL (at concentrations of 60, 90, 120, and 150 μM) preserved cellular morphology and improved barrier function. It achieved this by upregulating tight junction proteins such as ZO-1 and Occludin and significantly suppressing the secretion of inflammatory cytokines, including TNF-α, IL-1β, IL-6, IL-8, and IL-12. Additionally, GL downregulated mRNA expression of NF-κB and related pro-inflammatory mediators, reinforcing its role in maintaining mucosal integrity under inflammatory stress [41].

Further, a study examining both GL and isoliquiritigenin, bioactive compounds derived from Glycyrrhiza uralensis, showed that GL exerted a dose-dependent inhibitory effect on IL-6 production in LPS-treated macrophages. Mechanistically, it blocked the formation of the LPS–TLR4/MD-2 complex and prevented TLR4 homodimerization, thus modulating the initial stages of the innate immune response [40]. In addition to these upstream effects, GL also disrupts critical downstream mechanisms of inflammation and sepsis. It has been shown to competitively bind to HMGB1, thereby preventing HMGB1 from mediating the cytosolic delivery of LPS, an essential step for caspase-11 activation. This inhibition impairs the pyroptotic pathway and reduces systemic inflammation, coagulopathy, and lethality associated with sepsis. In vivo studies confirmed that GL reduced organ damage, cytokine release, and mortality in models of endotoxemia and septic shock [13].

2.1.11. Effect on Thermal and Burn Injury

Burn injury often results in systemic inflammation and organ damage. GL, known for its anti-inflammatory properties, has shown significant potential in protecting against burn-induced systemic inflammation. In a rat model of thermal injury, GL reduced the levels of pro-inflammatory cytokines (TNF-α, IL-1β), decreased HMGB1 mRNA and protein levels, and ameliorated injury in distant organs, such as the liver and lungs. This suggests that GL acts as an effective anti-inflammatory agent following thermal injury [42]. Mitochondrial dysfunction is a critical consequence of burn injury, leading to muscle wasting and weakness. Fibrinogen, a DAMPs protein, contributes to inflammation and mitochondrial dysfunction in skeletal muscles. GL administration alleviates burn-induced mitochondrial dysfunction by suppressing fibrinogen’s effects on inflammatory responses and mitochondrial membrane potential loss in muscle cells. This effect improves muscle integrity and survival rates in burn injury models [43].

2.1.12. Effect on Inflammation and Cytokine Expression

GL has shown potent anti-inflammatory effects in various models of systemic inflammation, including LPS-induced endotoxemia, periodontal disease, and mastitis. In endotoxemia, a model of systemic inflammation, GL significantly improved survival rates in LPS-treated mice and reduced the production of key inflammatory cytokines such as TNF-α, IL-6, IL-1β, and RANTES. In vitro, GL inhibited the production of these cytokines in LPS-stimulated RAW264.7 cells. Its mechanism of action involves the inhibition of NF-κB and IRF3 signaling pathways, along with disruption of lipid rafts. This disruption prevents the translocation of TLR4, thereby blocking the activation of downstream inflammatory cascades [44].

In periodontal disease models, GL also demonstrated significant anti-inflammatory effects. In LPS-stimulated human gingival fibroblasts, GL reduced the production of pro-inflammatory cytokines (IL-6, IL-8) and enzymes such as COX-2 and iNOS. These effects were mediated through the activation of liver X receptor alpha (LXRα) [45]. Similarly, in a mouse model of mastitis induced by LPS, GL reduced histopathological damage, inhibited neutrophil infiltration, and decreased the expression of inflammatory cytokines (TNF-α, IL-1β, IL-6). In vitro studies confirmed that GL suppressed LPS-induced activation of NF-κB and IRF3. These effects were attributed to GL’s ability to activate ABCA1, a protein that facilitates cholesterol efflux from lipid rafts, thereby inhibiting TLR4-mediated signaling [46].

2.1.13. Effect on Inflammatory Pain

Chronic inflammatory pain is often driven by microglial activation in the central nervous system. GL has been shown to suppress LPS-induced microglial activation and inflammatory cytokine production (IL-6, TNF-α, IL-1β) in BV2 microglial cells. Mechanistically, GL inhibits the HMGB1-TLR4-NF-κB signaling pathway, which is crucial for microglial activation. Additionally, GL alleviates inflammatory pain symptoms, such as mechanical allodynia and thermal hyperalgesia, in animal models [47]. In a study investigating the combined effects of GL, ligustrazine, and puerarin, GL showed significant anti-inflammatory activity by inhibiting the expression of 23 inflammatory cytokines in LPS-stimulated RAW264.7 cells. This multi-targeted approach highlights GL’s potential in modulating complex inflammatory networks [48].

2.1.14. Effect on Radiation-Induced Tissue Damage

GL mitigates radiation-induced tissue damage through anti-inflammatory and antioxidant mechanisms. In a murine model of radiation enteritis, C57BL/6 mice were subjected to 6.5 Gy abdominal X-ray irradiation to induce intestinal injury. GL treatment significantly improved histological architecture of the jejunum and reduced serum levels of pro-inflammatory cytokines (TNF-α, IL-6, IL-1β, and HMGB1). Furthermore, GL downregulated the HMGB1/TLR4 signaling pathway, a key mediator of inflammation in radiation-induced intestinal damage [49]. In another study, a rat model of radiation-induced salivary gland injury, GL was found to protect against radiation sialadenitis by acting as a mitochondria-targeted antioxidant. GL administration suppressed oxidative stress markers such as malondialdehyde and glutathione disulfide (GSSG), enhanced antioxidant enzyme activities (SOD, CAT, GPx), increased the GSH/GSSG ratio, and preserved mitochondrial structure and function. Mechanistically, GL downregulated HMGB1/TLR5 signaling and activated the PGC-1α/NRF1/NRF2/TFAM pathway, supporting mitochondrial biogenesis and function [50].

2.1.15. Effects Against Reproductive and Autoimmune Inflammatory Conditions

Effect on Endometriosis: Endometriosis is a chronic inflammatory condition where endometrial-like tissue grows outside the uterus, leading to pelvic pain and infertility. GL has shown promising anti-inflammatory effects in models of endometriosis. In one study, GL treatment of LPS-stimulated mouse endometrial epithelial cells (MEEC) resulted in the suppression of key inflammatory mediators, including TNF-α, IL-1β, NO, and PGE2. Additionally, GL reduced the expression of pro-inflammatory enzymes such as COX-2 and iNOS, along with key receptors like TLR4 and transcription factors like NF-κB. These findings suggest that GL may modulate inflammation through the TLR4/NF-κB signaling pathway, making it a potential therapeutic candidate for managing endometriosis-related inflammation [51].

Effect on Systemic Sclerosis: Systemic sclerosis (SSc) is an autoimmune inflammatory disorder that leads to fibrosis, vasculopathy, and immune dysregulation. GL has shown promise in alleviating several key aspects of this disease. In bleomycin-induced mouse models of SSc, GL reduced dermal fibrosis by inhibiting TGF-β signaling in fibroblasts and downregulating pro-fibrotic factors like thrombospondin-1 (TSP-1) and Smad3. Additionally, GL suppressed immune polarization toward a Th2 phenotype, reduced the infiltration of M2 macrophages, and decreased endothelial-to-mesenchymal transition (EndMT), which is a key process in fibrosis [52].

Effect on Preeclampsia: GL served as a promising therapeutic agent for managing preeclampsia through anti-inflammatory and HMGB1-modulating mechanisms. GL (60 mg/kg/day) significantly ameliorates preeclampsia symptoms in a rat model by reducing blood pressure, proteinuria, and improving pregnancy outcomes. It effectively suppresses systemic and placental inflammatory cytokines (TNF-α, iNOS, IL-1, IL-6) and downregulates the HMGB1/TLR4 signaling pathway in L-NAME-induced preeclampsia in pregnant rats [53].

2.1.16. Effects Against Colitis and Mucosal Inflammation

Effect on Ulcerative Colitis: Several studies have explored the therapeutic potential of GL in treating ulcerative colitis (UC) and experimental colitis, focusing on its anti-inflammatory effects and underlying mechanisms. In a murine model of colitis induced by oxazolone, mice were treated with 20 mg/kg/day of compound GL for 7 days. The results showed significant reductions in the disease activity index (DAI), macroscopic and microscopic injury scores, and MPO activity. Additionally, GL inhibited the activation of NF-κB and STAT3 signaling pathways [54]. Kudo et al. [55] assessed the efficacy of a GL preparation (GL-p) in rats with colitis induced by dextran sodium sulfate (DSS). Rats were treated with 20 mg/kg/day of GL-p intrarectally for 7 days. GL-p significantly improved the extent of colitis, as evidenced by reduced colon wet weight, improved macroscopic damage scores, and decreased MPO activity. The treatment also reduced levels of pro-inflammatory cytokines and chemokines, including IL-1β, IL-6, TNF-α, and monocyte chemoattractant protein-1.

In another study by Xu et al. [56], GL was administered at 20 mg/kg/day for 14 days in a rat model of TNBS-induced colitis. This treatment significantly improved disease activity, reduced macroscopic and histological damage, and lowered the expression of NF-κB p65 and iNOS in the colonic mucosa. Furthermore, GL treatment elevated the serum level of the anti-inflammatory cytokine IL-4 while reducing the pro-inflammatory cytokine IL-8. Moreover, Chen et al. [57] examined the effects of GL in a TNBS-induced colitis model in mice, with 20 mg/kg/day of GL administered for 14 days. The results showed that GL reduced the production of inflammatory mediators such as HMGB1, TNF-α, IL-6, and IL-17, and suppressed Th17 cell proliferation. Additionally, GL regulated the function of dendritic cells and macrophages, both of which play critical roles in immune responses during colitis.

In a clinical study, Sethuraman et al. [58] evaluated the combination of GL (administered at 20 mg/kg/day) and emu oil in an acetic acid-induced rat model of UC. This combination therapy significantly reduced both macroscopic and microscopic lesions, decreased MPO activity, and improved antioxidant enzyme levels. Furthermore, the combined treatment modulated the expression of PPARγ and TNF-α, with a more pronounced effect observed in the combined treatment group.

Effect on Mouth Ulcers: A water-soluble GL-based gel formulation of triamcinolone was found to be as effective as the standard Kenalog in Orabase for treating recurrent aphthous stomatitis in a clinical study with 134 patients. Its improved taste and texture offer better patient compliance. Additionally, GL alone provided pain relief and accelerated healing [59].

2.1.17. Effect Against Granulomatous Inflammation

Granuloma formation, often seen in chronic inflammatory diseases like sarcoidosis, is driven by excessive immune cell activation and fibroblast proliferation. In a guinea pig model of pulmonary granulomas induced by Sephadex beads, GL suppressed granuloma formation by inhibiting both fibroblast activation and monocyte stimulation. These results suggest that GL may offer therapeutic benefits in diseases characterized by granulomatous inflammation, potentially reducing the formation and progression of granulomas in conditions such as sarcoidosis [60].

2.2. Effect of Glycyrrhizin on Central Nervous System

Glycyrrhizin’s neuroprotective capacity is primarily attributed to its metabolite, 18β-glycyrrhetinic acid, which counteracts HMGB1. This mechanism has gained significant attention for its potential in treating various neurological disorders, including traumatic brain injury, neuroinflammation, epileptic seizures, Alzheimer’s disease, Parkinson’s disease, and multiple sclerosis. By neutralizing HMGB1, GL exhibits potential in slowing the progression of these conditions. Its neuroprotective effects stem from its ability to suppress HMGB1 expression and inhibit its translocation, leading to a reduction in inflammatory cytokine levels. This mechanism holds the potential to decelerate the advancement of neurological diseases by alleviating the inflammatory response.

2.2.1. Effect Against Intracerebral Hemorrhage

The therapeutic potential of GL, an inhibitor of HMGB1, for intracerebral hemorrhage (ICH) has been explored in previous studies. While investigating its impact, GL at concentrations of 10–100 μM exhibited a concentration-dependent protective effect against thrombin-induced cortical injury in rat cortico-striatal slice cultures. This protective effect was reversed by the application of exogenous HMGB1. Moreover, in a rat ICH model, GL (50 mg/kg) administration demonstrated beneficial effects. It attenuated ICH-induced edema in the cortex and basal ganglia, improved behavioral performance, and partially ameliorated neuron loss within the hematoma [61].

2.2.2. Effects Against Neuronal Cell Damage

GL may serve as protective agents against neuronal cell damage caused by 7-ketocholesterol by preserving the integrity of mitochondrial membrane permeability. At different concentrations ranging between 20 μM and 100 μM, it exhibited protective effects against the toxicity of 7-ketocholesterol, particularly concerning mitochondria-mediated cell death. When exposed to 7-ketocholesterol, differentiated PC12 cells experienced harmful effects such as nuclear damage, loss of mitochondrial transmembrane potential, increased levels of cytosolic Bax and cytochrome c, caspase-3 activation, and ultimately cell death. However, GL demonstrated the ability to prevent these adverse mitochondrial effects induced by 7-ketocholesterol. Consequently, it hindered caspase-3 activation and protected against cell death [62]. In a rat model with subacute neuroterminal norepinephrine depletion induced by fusaric acid, it exhibited neuroprotective effects. Despite not completely restoring norepinephrine levels, it mitigated imbalances in neurotransmitters, reduced inflammatory tissue damage, and enhanced bioenergetic status at dosage of 100 mg/kg for 30 days. Additionally, high doses of GL improved behavioral changes, minimized oxidative damage, and reduced apoptotic markers induced by fusaric acid including alleviation of GABA and histamine [63].

2.2.3. Effects Against Ischemia–Reperfusion-Induced Brain Injury

GL has demonstrated significant neuroprotective effects against cerebral I/R injury through multiple mechanisms, notably by inhibiting inflammation, oxidative stress, and apoptotic pathways. In a rat model of transient middle cerebral artery occlusion, subcutaneous administration of GL (100 mg/kg) for three consecutive days prior to ischemia suppressed lipid peroxide elevation in cerebral tissue and serum and enhanced superoxide dismutase (SOD) levels, indicating potent antioxidant activity [64]. Similarly, it significantly reduced infarct volume, improved neurological deficits, and alleviated apoptotic injury in I/R rats. These protective effects were linked to the inhibition of HMGB1 release from the cerebral cortex and downregulation of pro-inflammatory and oxidative markers such as TNF-α, iNOS, IL-1β, and IL-6. The study also highlighted the involvement of P38 and P-JNK signaling pathways in GL’s action [65]. In related studies, GL exhibited neuroprotection by modulating the HMGB1-TLR4-IL-17A signaling pathway, reducing both HMGB1 and IL-17A expression in TLR4-mutant mice. It significantly reduced brain injury and neurological deficits following ischemia [66]. Wagle et al. [67] found that it inhibited BACE1 with an IC50 of 20.12 µM, comparable to quercetin (IC50 = 20.18 µM), further supporting its role in protecting neuronal tissue.

Xiong et al. [68] demonstrated that GL’s efficacy was evident only in T cell–reconstituted animals, confirming its role in inhibiting HMGB1 release and T cell proliferation. Consistent findings from other ischemic models support GL’s ability to reduce brain injury by attenuating HMGB1-mediated inflammation and oxidative stress.

Clinically, Chen et al. [69] linked elevated peroxynitrite (ONOO⁻) and HMGB1 levels to hemorrhagic transformation in ischemic stroke patients undergoing delayed t-PA therapy. Complementary animal studies showed that ONOO⁻ directly activates HMGB1, triggering MMP-9–mediated blood–brain barrier disruption. GL effectively suppressed ONOO⁻ production and inhibited the ONOO⁻/HMGB1/TLR2 axis, reducing HT and improving neurological outcomes.

Similarly, in a rat model of FeCl₃-induced cerebral venous sinus thrombosis followed by mechanical thrombectomy, GL administered post-recanalization reduced infarct size and cerebral edema. Its protective effects were associated with inhibition of HMGB1 translocation, downregulation of RAGE expression, and attenuation of oxidative stress and inflammatory responses, leading to enhanced recovery [70].

2.2.4. Effects Against Traumatic Brain Injury

In a traumatic brain injury (TBI) rat model, intravenous glycyrrhizin (GL, 10 mg/kg) significantly reduced brain edema and motor deficits within 24 h post-injury. These neuroprotective effects were associated with inhibition of HMGB1 and its receptors (TLR4, RAGE), suppression of NF-κB activation, and downregulation of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6). GL also dose-dependently reduced blood–brain barrier permeability and improved motor and cognitive outcomes for up to 7 days post-injury [71,72].

Similarly, in a vascular dementia model induced by bilateral common carotid artery occlusion, GL (20 mg/kg for 5 days) markedly improved spatial learning and memory performance and alleviated long-term potentiation impairment. These benefits were accompanied by reduced hippocampal and cortical lipid peroxidation, enhanced superoxide dismutase activity, and decreased neuronal damage. Additionally, in vitro studies demonstrated that GL (10–50 μM) inhibited voltage-gated sodium channel currents in CA1 pyramidal neurons [73].

2.2.5. Effects Against Post-Traumatic Stress Disorder

Post-traumatic stress disorder (PTSD) has emerged as a significant health concern, with neurotransmitters and the amygdala playing crucial roles in its development and persistence. Impaired fear memory extinction is a central feature of PTSD, which may be driven by neuroinflammation resulting from single prolonged stress (SPS) exposure. Examining HMGB-1 levels in the basolateral amygdala (BLA) post-SPS, along with assessing microglia and astrocyte activation, reveals that intra-BLA administration of GL can prevent SPS-induced fear extinction deficits. Notably, GL treatment not only targets short-term HMGB1/TLR4-mediated pro-inflammation but also influences microglial and astrocytic responses to fear-related stimuli [74]. Moreover, GL treatment significantly alleviated anxiety and fear memory at 1 and 7 days post-PTSD induction. It also restored the disrupted circadian rhythm of serotonin and TPH2 expression. The observed restoration of serotonin diurnal fluctuations in the amygdala further underscores the promising role of GL in PTSD management [75].

2.2.6. Effects Against Sciatic Nerve Injury

While investigating the effects of GL on sciatic nerve injury in a mouse model, its oral administration at doses of 10 and 20 mg/kg per day was found to inhibit the expression of the p75 neurotrophin receptor (p75NTR) and improve nerve function following injury [76]. Additionally, higher doses of GL promoted better myelination of the sciatic nerve and reduced local scar formation and inflammation. The molecule can also enhance sciatic nerve regeneration and functional recovery, with higher doses showing greater effectiveness. This regeneration is associated with the downregulation of p75NTR.

2.2.7. Effects Against Neuro-Toxicity

Earlier research suggests that GL may protect against neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced brain tissue damage by inhibiting oxidative stress and alleviate 1-methyl-4-phenylpyridinium (MPP+)-induced toxicity by suppressing caspase-3 activation, likely due to its antioxidant property. In Parkinson’s disease (PD), HMGB1 may link progressive dopaminergic degeneration and chronic neuroinflammation, key components of the disease’s pathophysiology. Studies on PD patients and a mouse model induced by MPTP demonstrate elevated HMGB1 levels. Neutralizing HMGB1 antibodies in mice partially prevent dopaminergic cell death and reduce RAGE and TNF-α levels. GL binds to HMGB1, decreases MPTP-induced HMGB1 and RAGE expression, leading to reduced dopaminergic cell death. These findings suggest HMGB1 as a potential therapeutic target for neuroprotective strategies in PD [77]. It also demonstrates neuroprotective effects against PD in a MPTP-induced zebrafish model. GL co-treatment restored dopaminergic neuron length, reduced apoptosis, and improved locomotor activity. It also prevented vascular damage and downregulated neuroinflammatory genes (HMGB1A, TLR4B, NFkB, IL1Β, IL6) while modulating autophagy-related genes, suppressing α-syn and atg5, and upregulating parkin and pink1. Molecular docking confirmed GL’s binding to HMGB1, TLR4, and RAGE [78]. GL attenuated MPTP-induced elevated brain enzyme activities and oxidative stress markers in mice. In vitro, it reduced MPP+-induced cell death and caspase-3 activation in PC12 cells, showing efficacy at concentrations up to 100μM. Additionally, it mitigated cell death induced by hydrogen peroxide or nitrogen species [79].

Boshra et al. [80] investigated the protective effects of GL and mangiferin against aluminum-induced brain toxicity in rats and compared their efficacy to that of melatonin. Various parameters related to brain health, including lipid profile, levels of proteins associated with apoptosis (Bcl2 and Bax), antioxidant enzymes (GSH, SOD, CAT), markers of oxidative stress (malondialdehyde, NO), and inflammatory cytokines (TNF-α, IL-22, and IL-1β), together with the gene expression of brain antioxidant enzymes (SOD, CAT) and interleukin 22 (IL-22), were evaluated. The treatment with GL and mangiferin, either alone or in combination, as well as melatonin co-treatment before aluminum chloride intoxication, significantly improved all biochemical parameters relevant to brain function. This improvement in brain function in aluminum-intoxicated rats was attributed to the antioxidant and anti-inflammatory properties of these compounds.

2.2.8. Effects Against Status Epilepticus and Seizures

GL has been shown to exert neuroprotective effects in various models of brain injury, including postischemic brain damage and kainic acid injury in rats. By scavenging reactive oxygen species and suppressing oxidative stress, GL mitigates neuronal damage and maintains cellular integrity. Moreover, GL (50 mg/kg, i.p.) inhibits the release of pro-inflammatory cytokines, such as interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α), thereby reducing neuroinflammation in the lithium/pilocarpine-induced seizure model of rats in hippocampus and olfactory bulb [81]. By blocking HMGB1 release and its translocation from the nucleus to the cytoplasm, GL protected against neuronal damage and maintained blood–brain barrier integrity in conditions such as status epilepticus induced by lithium-pilocarpine in rats. This molecule may be a valuable therapeutic agent for neurologic disorders characterized by inflammation and neuronal injury [82]. In the kainic acid (KA)-induced seizure model, GL administered prior to KA reduced neuronal cell death, suppressed inflammation, and inhibited HMGB1 induction and release, particularly in the hippocampal CA1 and CA3 regions. HMGB1 expression peaked at 3 h and 6 days post-KA, initially in neurons and astrocytes, later accumulating in serum. GL’s neuroprotective effects in this model likely stem from its ability to curb HMGB1-related inflammation and cell death [83]. In KA-induced epilepsy in rats, GL, when administered intraperitoneally, reduced seizure incidence (50%) and mortality (10%) compared to KA alone (80% and 40%, respectively). It attenuated hippocampal neuronal damage, preserved Neu-N-positive neuronal cells, and suppressed mRNA and protein expression of HMGB1, TLR4, and phosphorylated NF-κB, indicating its protective role through inhibition of the HMGB1/TLR4/P-NF-κB inflammatory signaling pathway [84]. In neurological models, GL (25, 50, and 100 mg/kg, i.p.) conferred anticonvulsive effects in zebrafish with pentylenetetrazol-induced chronic seizures. It significantly improved behavior, memory, and seizure outcomes while downregulating the HMGB1-TLR4-NF-κB axis and modulating key neuroplasticity genes such as BDNF, CREB-1, and NPY [85].

2.2.9. Effects Against Neuroinflammation and Cognitive Deficits

GL has the potential to attenuate isoflurane-induced neuroinflammation, prevent spatial memory deficits, and alleviate neuroapoptosis in the hippocampus of neonatal rats [86]. When administered intraperitoneally to 7-day-old rats 30 min before exposure to 1.8% isoflurane, GL mitigated the increase in pro-inflammatory cytokines (TNF-α and IL-1β) and the activation of the HMGB1/NFκB signaling pathway in the neonatal rat hippocampus. This effect was accompanied by down-regulations of PSD-95 and SNAP-25. In addition, the treatment prevented the spatial memory deficits induced by neonatal exposure to isoflurane.

It has been observed that GL mitigates LPS-induced memory deficits by inhibiting pro-inflammatory mediators and microglial activation [87]. In C57BL/6 mice treated with systemic LPS, GL significantly reduces mRNA levels of TNF-α and IL-1β, as well as protein expressions of COX-2 and iNOS at doses of 30 and 50 mg/kg. In the Morris water maze test, administration of GL at 30 mg/kg improves swimming time in target and peri-target zones, along with target heading and memory score numbers. Additionally, it diminishes the up-regulated Iba1 protein expression and average cell size of Iba1-expressing microglia in hippocampal tissue induced by LPS.

In the case of surgery-induced neuroinflammation, the oral pretreatment of GL prevented postoperative cognitive dysfunction by inhibiting HMGB1-induced neuroinflammation and Alzheimer’s-related pathology in aged mice [88]. In aged male C57BL/6 mice undergoing splenectomy surgery, oral pretreatment with GL effectively inhibited HMGB1 cytosolic expression, increased PSD-95 protein expression, and mitigated postoperative memory impairment. Furthermore, its pretreatment reduced postoperative neuroinflammation, indicated by decreased production of pro-inflammatory cytokines (IL-1β, TNF-α, and IL-6) and NF-κB nuclear expression, along with Alzheimer’s-related pathology in the hippocampus, including reduced Tau phosphorylation at the sites of AT-8 and Ser396, as well as decreased concentrations of Aβ40 and Aβ42.

It showed protective effects in a necrotizing enterocolitis (NEC) mouse model by inhibiting the HMGB1/TLR4 pathway. It significantly reduced microglial pyroptosis and preserved the integrity of the blood–brain barrier and myelin basic protein, contributing to improved neurological outcomes [89]. Similarly, in models of status epilepticus in rats, GL decreased hippocampal neuronal damage and suppressed HMGB1 and p38MAPK signaling, thus preventing HMGB1 translocation and associated neuroinflammation [90].

2.2.10. Effects Against Subarachnoid Hemorrhage

In experimental subarachnoid hemorrhage (SAH), increased levels of Toll-like receptors (TLRs) and downregulation of peroxisome proliferator-activated receptors (PPARs) are observed. GL administration initiated 24 h before and 1 h after SAH induction to rats reduced morphological abnormalities in basilar arteries and attenuated TLR-2 and -4 expressions by 28% and 33.4%, respectively. Additionally, GL reduced IL-1β and MCP-1 levels, demonstrating its anti-inflammatory effects on SAH-induced vasospasm and early brain injury [91]. Similarly, in another rat SAH model, GL (10 mg/kg i.p. once daily for 3 days) significantly improved neurological function, increased basilar artery diameter, and reduced vascular wall thickness. Mechanistically, GL inhibited HMGB1 expression, downregulated pro-inflammatory cytokines (IL-1β, IL-6, TNF-α), and upregulated IL-10 in the basilar artery, thereby attenuating cerebral vasospasm and post-SAH inflammation [92].

2.2.11. Effects Against Anxiety and Stress

GL (100 and 200 mg/kg) exhibited notable antistress potential, demonstrated by its ability to reverse behavioral and biochemical changes induced by chronic immobilization stress in mice [93]. Furthermore, investigations into its antidepressant-like effects revealed promising outcomes. Administered to young male Swiss albino mice over seven days, GL at doses of 1.5, 3.0, and 6.0 mg/kg intraperitoneally reduced immobility periods [94]. Notably, the antidepressant-like effect was comparable to that of imipramine and fluoxetine. Additionally, GL did not significantly affect locomotor activity. The involvement of adrenergic and dopaminergic systems in its antidepressant-like effect was suggested, as evidenced by attenuation with sulpiride and prazosin, selective D2 receptor and α1-adrenoceptor antagonists, respectively, while p-chlorophenylalanine, an inhibitor of serotonin synthesis, had no effect. These findings imply that GL’s antidepressant-like mechanism may primarily involve the modulation of brain norepinephrine and dopamine levels.

2.2.12. Effects on Intracellular Calcium Mobilization and Neuromuscular Transmission

GL was found to affect Ca²⁺ mobilization in nerve-stimulated skeletal muscle of mice. At concentrations of 0.3–1 mM, GL depressed contractile Ca²⁺ transients without affecting non-contractile ones, indicating a potential to block neuromuscular transmission. Combined treatment with paeoniflorin (25 µg/mL) and GL (75 µg/mL) blocked muscle twitch tensions, showing a postsynaptic mechanism. Iontophoretically injected acetylcholine potential amplitudes were substantially inhibited by the combined treatment. Caffeine-induced contractures were reduced with GL treatment. Interestingly, this combination, blocked intracellular Ca²⁺ movement in muscles with sustained depolarization [95,96].

2.2.13. Effects Against Virus-Born Neurological Disorders

GL demonstrated protective effects against herpetic encephalitis induced in mice via corneal inoculation with herpes simplex virus 1 (HSV-1). Administered intraperitoneally to mice afflicted with herpetic encephalitis, GL significantly increased their survival rate by approximately 2.5 times and reduced HSV-1 replication in the brain to 45.6% of the control levels [97]. Additionally, autoimmune encephalomyelitis, characterized by immune-mediated inflammation and demyelination in the CNS, involves the release of the pro-inflammatory protein HMGB1. GL can mitigate the pro-inflammatory actions of HMGB1 [98]. Furthermore, a clinical case involving a 9-year-old girl diagnosed with subacute sclerosing panencephalitis exhibited improvement in motor and mental functions after receiving a treatment regimen including high-dose GL alongside anticonvulsants and recombinant IL-2. Notably, immunological abnormalities observed in the patient, such as reversed CD4/CD8 ratio and low NK cell activity, returned to normal levels following treatment, indicating the potential efficacy of GL in managing neurological disorders [99].

2.2.14. Effect Against Neonatal Hypoxic–Ischemic Brain Damage

In hypoxic–ischemic brain damage (HIBD) models, GL suppressed neuronal ferroptosis by downregulating HMGB1 and upregulating GPX4. Using RSL3 (a ferroptosis agonist) and oxygen-glucose deprivation models in vitro, and HIBD in vivo, GL was shown to inhibit ferroptosis-associated ultrastructural changes, oxidative stress, mitochondrial damage, and inflammatory gene expression. Mechanistically, GL exerted neuroprotection by modulating the HMGB1/GPX4 axis, reducing ROS and neuronal loss [100].

2.3. Effect of Glycyrrhizin on Respiratory System

GL emerges as a promising solution for respiratory issues, offering relief from rapid shock triggered by LPS and reducing pulmonary platelet accumulation, especially beneficial in acute respiratory distress syndrome (ARDS) linked with sepsis. It effectively improves the severity and duration of upper respiratory tract infections (URTIs) in hospitalized patients without acute bacterial infections, showcasing its tolerability, efficacy, and cost-effectiveness. Additionally, GL demonstrates potential in mitigating S. aureus-induced acute lung injury (ALI) by modulating inflammation and immune pathways [101,102,103,104,105,106,107,108,109,110,111]. It also protects against LPS-induced ALI and shows promise in addressing sepsis-induced organ damage, while exhibiting anti-inflammatory properties in carrageenan-induced lung injury [112].

2.3.1. Effect Against Severe Acute Respiratory Syndrome

GL administration in mice inhibited platelet responses and decreased delayed lethality in LPS-induced rapid shock. Given intravenously at a dosage of 200 mg/kg, GL reduced pulmonary platelet accumulation and mitigated the severity of LPS-induced rapid shock, preventing mortality both shortly after and later on. At moderate doses, it exhibited no adverse effects. It has the potential to alleviate acute respiratory distress syndrome associated with sepsis and demonstrate possible efficacy against severe acute respiratory syndrome [101]. In an in vivo study, Gu et al. [102] also found that GL improves survival and lung function in a mouse model of sepsis-induced ARDS. It reduced lung tissue injury, protein leakage, and inflammation by inhibiting neutrophil extracellular trap formation through suppression of the HMGB1/TLR9/MyD88 signaling pathway.

2.3.2. Effect Against Upper Respiratory Tract Infections

A clinical study conducted at the Japanese Maritime Self-Defense Force Etajima Hospital revealed that GL therapy resulted in reduced hospital stays, decreased fever intensity, and lower treatment costs for patients with URTIs. Patients received an intravenous drip infusion of 40 mL of GL (0.2%) and 500 mL of lactated Ringer’s solution daily during hospitalization. The study included a total of 41 patients, with 15 receiving GL treatment and 26 assigned to the control group, who received only an intravenous drip infusion of 500 mL/d of lactated Ringer’s solution [103].

2.3.3. Effect Against Acute Lung Injury

Several studies have investigated the potential therapeutic effects of GL on various models of ALI induced by different triggers. It has been found that GL holds promise as a therapeutic agent for ALI and related conditions by exerting anti-inflammatory effects, suppressing immune responses, and modulating signaling pathways involved in inflammation and tissue injury. In LPS-induced ALI models, GL has shown consistent anti-inflammatory and protective effects. GL treatment at a dose of 20 mg/kg reduced inflammation by downregulating the expression of NF-κB mRNA and TNF-α mRNA, while upregulating GR mRNA expression and IL-10 protein levels in rats. It also attenuated lung injury histopathologically and reduced the infiltration of polymorphonuclear cells [104]. Similarly, GL at doses of 10, 25, and 50 mg/kg decreased protein content, inflammatory cell counts, MPO activity, and expressions of COX-2, iNOS, and NF-κB, while also inhibiting the migration and infiltration of inflammatory cells and downregulating TLR4 expression [105,106]. In another study, GL (4, 8, 16 mg/kg, i.p.) alleviated lung injury by decreasing TNF-α and IL-1β levels and increasing IL-10 levels, along with suppressing the expression of Bax and promoting the expression of Bcl-2 in lung tissues [107]. Furthermore, it demonstrated protective effects on the heart and lungs in LPS-induced septic mice by increasing the ratio of MDSCs to CD11b + Gr1 myeloid cells and inhibiting cytokine release and TLR4 expression [108].

Recent studies have further elaborated GL’s molecular mechanisms in ALI. GL was shown to alleviate LPS-induced ALI both in vitro (RAW 264.7 macrophages) and in vivo (BALB/c mice) by reducing inflammatory mediators (NO, PGE2, IL-1β, IL-6, TNF-α, IL-18) and inhibiting NF-κB activation and NLRP3 inflammasome signaling. High-dose treatment of GL showed comparable efficacy to dexamethasone in reducing lung inflammation, MPO activity, and lung edema [109]. Bioinformatics analysis combined with in vivo experiments further suggested that GL suppressed the TLR signaling pathway, particularly by inhibiting TLR2, thereby reducing the expression of downstream inflammatory mediators and attenuating lung inflammation and pathological changes [110]. In a model of S. aureus-induced ALI, GL (25 mg/kg, i.p.) alleviated inflammation by reducing multiple cytokines and immune cells and inhibiting inflammatory and pyroptotic signaling pathways. It reduced levels of IL-6, TNF-α, IL-8, IL-1β, and HMGB1, while suppressing NF-κB and p38/ERK pathways. In vitro experiments indicated that the p38 pathway was the primary route for S. aureus-induced inflammation [111].

GL also demonstrated a protective effect in I/R-induced lung injury. Pretreatment with 200 mg/kg, i.p., ameliorated pulmonary permeability and edema in mice, inhibited I/R-induced inflammation in lung tissues and bronchoalveolar lavage fluid, and reduced alveolar macrophage counts. It also suppressed the expression of TLR2 and downstream factors in lung tissues and macrophages [112]. In radiation-induced lung injury models, GL reduced lung inflammation and HMGB1 and pro-inflammatory cytokine levels by inhibiting the HMGB1/TLR4 signaling pathway [113]. It also exerted radioprotective effects by inhibiting ER stress and NLRP3 inflammasome activation, as shown in irradiated mice and MLE-12 cells. Systemic pharmacology suggested involvement of the MAPK signaling pathway [114].

In carrageenan-induced pleurisy, GL (10 mg/kg, i.p.) attenuated fluid accumulation in the pleural cavity, PMN infiltration, lipid peroxidation, and pro-inflammatory cytokine production. It also reduced the expression of adhesion molecules and oxidative stress markers, and inhibited NF-κB and STAT-3 activation in lung tissues [115]. GL also protected against particulate matter-induced lung injury by activating the Nrf2/HO-1/NQO1 antioxidant pathway, thereby reducing oxidative stress, ER stress, and NLRP3 inflammasome-mediated pyroptosis in mice and human bronchial epithelial cells. The use of an Nrf2 inhibitor (ML385) confirmed the Nrf2-dependence of GL’s protective effects [116].

2.3.4. Effect Against Asthma and Chronic Obstructive Pulmonary Disease

GL has shown significant potential in managing asthma and airway inflammation through its anti-inflammatory, immunomodulatory, and mucoregulative properties. In a mouse model of asthma induced with ovalbumin (OVA), oral administration of GL at 5 mg/kg effectively inhibited airway constriction, methacholine-induced hyperreactivity, and lung inflammation. It significantly reduced IL-4, IL-5, and eosinophil levels in bronchoalveolar lavage fluid (BALF), as well as serum OVA-specific IgE levels, without altering serum cortisol [117]. Similarly, oral treatment at 10 mg/kg in a chronic asthma mouse model alleviated histopathological changes such as goblet and mast cell proliferation, epithelial thickening, and smooth muscle layer expansion. Notably, the effects were comparable to those of dexamethasone (1 mg/kg) [118].

In airway epithelial models, GL attenuated mucin 5AC (MUC5AC) overproduction induced by human neutrophil elastase in 16HBE cells via inhibition of the p38-NF-κB p65/IκBα signaling pathway [119]. Similar results were observed in NCI-H292 cells, where GL reduced MUC5AC gene expression and protein production induced by epidermal growth factor or phorbol ester [120]. In IL-13-stimulated rat models and HBE-16 cells, GL downregulated MUC5AC expression by mitigating oxidative stress, aldose reductase activity, and reactive oxygen species generation [121]. These findings align with those of Nishimoto et al. [122], where both in vivo and in vitro models demonstrated that GL reduced goblet cell hyperplasia and MUC5AC mRNA levels. Further clinical evidence of GL’s mucoregulatory effect was reported by Miyazaki et al. [123], where intravenous administration of 80 mg GL successfully controlled mucus overproduction in a patient with mucin-producing bronchoalveolar carcinoma, facilitating surgical intervention.

GL’s mechanism of action differs from that of traditional glucocorticoids. In A549 lung epithelial cells, both GL and dexamethasone suppressed IL-8 production induced by TNF-α and IL-1β, but only GL inhibited NF-κB p65 binding to the IL-8 promoter independently of glucocorticoid receptor pathways [124]. In human colonic epithelial Caco-2 cells, GL also enhanced the production and gene expression of secretory components [124]. Moreover, GL was shown to boost secretory component expression in airway models, resembling glucocorticoid activity but via distinct molecular mechanisms [125].

Protective effects of GL were also evident in studies investigating β2-adrenergic receptor (β2AR) internalization and apoptosis. It significantly inhibited salbutamol-induced β2AR internalization by reducing interactions with β-arrestins and clathrin heavy chain, and by attenuating GRK-mediated β2AR phosphorylation. This preserved β2AR mRNA and protein levels, enhancing receptor-mediated signaling. Additionally, GL mitigated salbutamol-induced early apoptosis via modulation of Bcl2 family gene expression [126]. It also inhibited TGF-β1-induced epithelial–mesenchymal transition (EMT) in A549 and BEAS-2B cells through HMGB1 suppression and Smad2/3 signaling blockade, thus reducing cell migration and fibrotic progression [127].

2.3.5. Effects Against Nitrogen Species-Mediated Lung Cell Damage

GL appears to mitigate nitrogen species-mediated lung cell damage by counteracting mitochondrial dysfunction and cell death in lung epithelial cells exposed to 3-morpholinosydnonime, a NO and superoxide donor. Treatment with GL attenuates 3-morpholinosydnonime-induced mitochondrial damage, ROS formation, and GSH depletion, exhibiting maximal inhibitory effects at concentrations of 10 and 1 μM, respectively. It appears to mitigate the toxic effects of 3-morpholinosydnonime on lung epithelial cells by suppressing mitochondrial permeability transition, which prevents the release of cytochrome c and the activation of caspase-3. This preventive effect may be attributed to its inhibitory action on reactive oxygen species formation and GSH depletion [128].

2.3.6. Effects Against Allergic Rhinitis

A clinical study by Cavone et al. [129] investigated the effects of a GL-containing formulation on HMGB1 levels in the nasal fluids of rhinitis patients. The treatment significantly reduced HMGB1 levels, comparable to nasal budesonide treatment, and selectively killed eosinophils without affecting other leukocytes. In allergic rhinitis (AR) mice, GL normalized lipid peroxidation and enhanced antioxidant defenses. It also increased IFN-γ and reduced IL-4 levels, thereby improving antioxidant status and immunity. Another study on AR mice induced by ovalbumin showed that GL significantly reduced IgE, IL-4, IL-5, IL-6, NO, TNF-α levels, and NOS activity, while increasing IgA, IgG, IgM, IL-2, and IL-12 levels. It also enhanced acetylcholinesterase activity and reduced substance P levels, indicating improved immune function [130]. Furthermore, GL inhibited histamine-induced expression and secretion of MUC5AC, IL-6, and IL-8 in human nasal epithelial cells. It restored AQP5 and p-CREB expression levels and inhibited NF-κB pathway activation [131].

2.4. Effect of Glycyrrhizin on Cardiovascular System

Previous research has demonstrated that GL can directly influence heart performance. It has been shown to significantly enhance heart muscle contraction (positive inotropic effect) and relaxation (positive lusitropic effect), even at low concentrations [132]. Additionally, GL markedly increased heart rate. These effects are mediated through the endothelin receptor type A/phospholipase C signaling pathway. In a study by Chen et al. [133], the effects of GL on vasospasm in chicken embryos after hemorrhage were investigated at the doses of 3 μg and 6 μg. The results showed that GL, particularly at the higher dose, improved survival rates and alleviated allantoic cavity artery (ACA) vasospasm. GL reduced oxidative stress, inflammation (IL-6, TNF-α), apoptosis (C-caspase-3, Bcl-2), and modulated MAPK pathways (p-P38/P38, p-JNK/JNK). The various effects of this molecule on the cardiovascular system are outlined below.

2.4.1. Anti-Hypertensive Effect

GL significantly increased systolic blood pressure in male Wistar rats treated with 200 mg/kg/day for 5 weeks. It enhanced pressor responses to norepinephrine and altered levels of vascular aldosterone and corticosterone. GL inhibited the expression of 11β-HSD2 and CYP11B2 mRNA in the mesenteric arteries, leading to lower aldosterone and higher corticosterone production, resulting in increased vasoconstrictor responses to norepinephrine [134]. In addition, it attenuated the progression of monocrotaline-induced pulmonary hypertension in rats, reducing right ventricular systolic pressure, right ventricular hypertrophy, and pulmonary inflammation. This was achieved through the inhibition of HMGB1 expression and improved survival rates in treated rats. These findings suggest GL’s potential as a therapeutic agent for pulmonary hypertension by inhibiting HMGB1 [135]. While exploring the impact of GL on vascular inflammation and atherosclerosis in Apoe−/− mice on a high-fat diet, Ding et al. [136] found that GL treatment (50 mg/kg for 12 weeks) reduced serum lipid levels, atherosclerotic plaque deposition, and serum HMGB1 levels. It also increased the Treg/Th17 ratio, elevated IL-10 and IL-2 expression, decreased IL-17A and IL-6 expression, reduced STAT3 phosphorylation in Th17 cells, and increased STAT5 phosphorylation in Treg cells.

GL has also demonstrated benefits in portal hypertension associated with liver cirrhosis. In bile duct ligation-induced cirrhotic rats, oral administration of GL (150 mg/kg/day) significantly reduced portal pressure, mesenteric blood flow, and portosystemic shunting by downregulating VEGF-mediated mesenteric angiogenesis [137]. Furthermore, GL exhibited protective vascular effects in a high-fat diet/streptozotocin-induced diabetic rat model. It attenuated endothelial senescence and restored vascular function by inhibiting HMGB1 expression and enhancing p53 activity both in vivo and in HUVECs [138].

2.4.2. Anti-Thrombotic Effect

GL has been identified as a thrombin inhibitor, prolonging plasma recalcification time and thrombin and fibrinogen clotting times. It inhibits thrombin-induced platelet aggregation but does not affect collagen-, PAF-, or convulxin-induced aggregation. GL interacts with thrombin’s anion-binding exosite 1 and does not block thrombin’s amidolytic activity on S-2238 but displaces hirudin as an inhibitor of thrombin-catalyzed hydrolysis of S-2238 [139].

Its intravenous administration significantly reduced thrombus size in a rat model of venous thrombosis. At doses of 180 mg/kg and 360 mg/kg, it decreased thrombus weight by 35% and 90%, respectively, and enhanced APTT ex vivo by 1.5- and 4.3-fold. However, doses above 90 mg/kg caused significant hemorrhagic effects and did not enhance heparin’s inhibitory activity towards thrombin [140]. Preoperative GL treatment was also found effective in preventing venous thrombosis by reducing neutrophil adherence to venous endothelium without increasing bleeding risk. Additionally, it reduced neutrophil binding to immobilized recombinant P-selectin in vitro [141].

2.4.3. Effect Against Myocardial Ischemia–Reperfusion Induced Injury

GL has shown protective effects against I/R-induced injuries in various organs of rats. The rats underwent a 30 min left coronary artery occlusion followed by 24 h of reperfusion and were treated with GL or GL plus recombinant HMGB1 after 30 min of ischemia. GL, administered intravenously at 10 mg/kg, significantly reduced infarct size and decreased serum levels of HMGB1, TNF-α, and IL-6. It also altered the distribution of Bax and cytochrome c between mitochondrial and cytosolic fractions in heart tissue, inhibiting myocardial apoptosis. Additionally, it decreased phospho-JNK expression, while co-administration with recombinant HMGB1 reversed these effects [142]. Its protective role in reducing I/R injury in rats was demonstrated through modulation of oxidative stress, iNOS, and inflammatory pathways involving HMGB1 and MAPK. Pre-treatment with GL significantly reduced infarct size and decreased the activities of creatine kinase, creatine kinase-MB, lactate dehydrogenase, and cardiac troponin T. Furthermore, it inhibited the expressions of phosphorylated ERK, p38 MAPK, and JNK proteins while promoting the expression of extracellular signal-regulated kinase [143]. Boissady et al. [144] found that early inhibition of HMGB1 using GL improves neurological outcomes after cardiac arrest in rabbits by reducing systemic inflammatory cytokines (IL-6, IL-10) and limiting cerebral infiltration of CD4⁺ and CD8⁺ T cells. These neuroprotective effects occurred independently of blood–brain barrier preservation and are replicated by blocking the HMGB1 receptor RAGE, showing HMGB1-RAGE signaling as a key therapeutic target for post-resuscitation brain injury.

2.4.4. Effect Against 3-Nitropropionic Acid-Induced Neurotoxicity

GL exhibits neuroprotective effects against 3-nitropropionic acid (3-NP)-induced neurotoxicity, an experimental model of Huntington’s disease (HD). Rats, pretreated with GL (50 mg/kg, i.p.) for 3 weeks, with 3-NP (10 mg/kg, i.p.) administered during the final two weeks to induce HD-like pathology, GL effectively countered 3-NP-induced neurobehavioral deficits, body weight loss, and neuronal damage. It attenuated oxidative stress by restoring antioxidant markers (GSH, SOD, Nrf2) and lowering MDA levels. It also inhibited neuroinflammation and apoptosis by downregulating the HMGB1/TLR4/NF-κB signaling pathway, reducing pro-inflammatory cytokines (IL-6, IL-1β, TNF-α), and caspase-3 expression, while upregulating anti-apoptotic Bcl-2 and neurotrophic BDNF [145].

2.4.5. Effect Against Coronary Microembolization-Induced Myocardial Dysfunction

In a rat model of coronary microembolization (CME), GL treatment significantly attenuated myocardial injury, as evidenced by reduced serum levels of cardiac troponin I and creatine kinase, and improvement in cardiac function parameters. It also inhibited the CME-induced increase in inflammatory mediators including HMGB1, TNF-α, iNOS, IL-6, and IL-1β. Moreover, it reduced cardiomyocyte apoptosis by downregulating cleaved caspase-3 and Bax expression while upregulating the anti-apoptotic marker Bcl-2. Mechanistically, these effects were attributed to the suppression of the HMGB1/TLR4/NF-κB signaling pathway, highlighting its dual role in modulating inflammation and apoptosis to protect against myocardial dysfunction [146].

2.4.6. Effect Against Viral Myocarditis

GL was found effective in alleviating Coxsackievirus B3 (CVB3)-induced myocarditis by improving weight loss, reducing serological levels of cardiac enzymes, diminishing myocardial inflammation, and increasing survival rates. The therapeutic effect was attributed to weakened pro-inflammatory responses rather than viral clearance, with GL reducing the expression of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6. Additionally, it inhibited CVB3-induced nuclear factor-κB activity by blocking the degradation of its inhibitor, IκBκ [147].

2.4.7. Effect Against Doxorubicin-Induced Cardiotoxicity

A study by Lv et al. [148] demonstrated that GL mitigates doxorubicin (DOX)-induced cardiotoxicity (DIC) in both in vitro (H9c2 cardiomyoblasts, neonatal rat cardiomyocytes) and in vivo (rat) models. DOX (20 mg/kg, i.p., single dose) induced cardiotoxicity as evidenced by elevated AST, CK-MB, oxidative stress, and disrupted mitochondrial membrane potential. GL (25 or 50 mg/kg/day, i.p., for 14 days) countered these effects. In vitro, GL (0.8 mM, 12 h) reduced DOX-induced cytotoxicity, oxidative damage, and improved mitochondrial stability. Mechanistically, GL restored autophagy flux impaired by DOX by modulating LC3-II and p62 levels, and inhibited the HMGB1/Akt/mTOR signaling pathway.

2.5. Effect of Glycyrrhizin on Various Liver Injuries and Conditions

GL exhibited hepatoprotective properties through various mechanisms, including enzyme suppression, gene regulation, and inhibition of oxidative stress and apoptosis pathways. At 1000 μg/mL, it was found effective in reducing liver damage induced by D-galactosamine and CCl₄ in rat models. Its high adsorbability in hepatocytes contributed to its strong protective effects against cytotoxicity [149]. It decreased plasma aspartate aminotransferase and alanine aminotransferase levels in chronic hepatitis patients. In rat hepatocytes, it suppressed the release of these enzymes induced by anti-liver cell membrane antibody and phospholipase A2 [150]. Moreover, In HepG2 cells, GL regulated genes involved in apoptosis, oxidative stress, and hepatocarcinogenesis. This molecule also inhibited NF-κB activity, contributing to its protective effects on liver cells [151]. GL was found to enhance hepatic differentiation from human iPS cells, especially during the hepatoblast stage. Mechanistically, GL-induced hepatic differentiation is mediated through the sweet taste receptor T1R3, as confirmed by the promotion by sucralose (T1R3 agonist) and inhibition by DP (T1R3 antagonist). Additionally, activation of Wnt/β-catenin signaling and suppression of Notch (Hes5) suggest these pathways contribute to the hepatic lineage commitment [152].

It significantly increased hepatic GSH content in isolated liver perfusion (43.7 μmol/L) as well as in intravenous treatment (25 mg/kg) in normal rats whereas a decrease in GSH biliary excretion was noticed after the treatment. The treatment also decreased the secretion rate of methotrexate in isolated liver perfusion and the ATP-dependent uptake of estradiol-17-β-glucuronide by Mrp2 membrane vesicles [153]. While pretreating isolated liver cells, GL substantially mitigated injuries inflicted by antibody-dependent cell-mediated cytotoxicity or LPS-induced activated macrophage culture supernatants. This indicates that GL also exerts a protective effect against immunological damage to liver cells [154]. It reduced liver inflammation and fibrosis, inhibited various T helper cell types, and enhanced antifibrotic cytokines IFN-γ and IL-10 in a concanavalin A (ConA)-induced mouse model. Additionally, it inhibited ConA-induced CD4⁺ T cell proliferation and related signaling pathways, thereby alleviating liver injury and fibrosis through immune modulation [155]. Various other protective effects of this natural molecule are outlined below.

2.5.1. Effect on Hepatectomy and Liver Regeneration

GL has significant hepatoprotective effects in various models of liver injury, through mechanisms involving antioxidation, anti-apoptosis, lipid metabolism regulation, and immune response modulation. It effectively enhanced liver regeneration and protected against liver injury, particularly under severe stress or damage from surgical resection. Administering GL (50 mg/kg/day, i.p.) significantly improved liver function and regeneration in 70% partially hepatectomized rats, as demonstrated by a higher liver weight to body weight ratio and increased DNA synthesis in liver cells. It also lowered elevated serum ALT and AST levels post-surgery [156]. Additionally, GL improved survival rates and reduced liver damage in rats exposed to endotoxins after hepatectomy. It decreased liver enzyme levels (AST, ALT, LDH), inhibited hepatocyte apoptosis by downregulating caspase-3 and preventing cytochrome C release, and reduced inflammation by inhibiting TNF-α, myeloperoxidase activity, and NF-κB translocation. Furthermore, GL promoted liver cell proliferation [157].

2.5.2. Effect on Liver Fibrosis and Hepatic Stellate Cell Activation

GL exhibited the ability to alleviate liver fibrosis through various mechanisms. These include the suppression of collagen gene expression, inhibition of hepatic stellate cell activation, induction of hepatic stellate cell apoptosis, and modulation of the TGF-β/Smad signaling pathway [158,159,160,161]. Notably, when combined with other compounds such as matrine or hinokiflavone, GL’s hepatoprotective effects were further potentiated [162,163]. These comprehensive studies underscore GL’s potential as a therapeutic avenue for liver diseases, offering a spectrum of benefits including antioxidative, anti-inflammatory, and hepatoprotective properties [164].

2.5.3. Effect on Hepatotoxins-Induced Liver Injury

GL can mitigate hepatotoxicity induced directly by hepatotoxins like carbon tetrachloride (CCl₄) and allyl formate. Pretreatment with GL 20 h before CCl₄ administration protected against pericentral hepatocellular necrosis. Similarly, pretreatment 2 h before allyl formate administration inhibited periportal hepatocellular necrosis [165]. In investigations into the hepatoprotective effects of GL against liver injury induced by various factors including CCl₄, ethanol, and endotoxin, significant findings emerged. In a study using LEC rats, an animal model for Wilson’s disease, subcutaneous administration of GL with a low-copper diet improved survival and weight gain. It shows its potential in preventing hepatic injury and liver cancer [166]. At a dosage of 100 mg/kg administered orally, GL demonstrated the capacity to mitigate liver injury, improve liver function, and impede fibrosis in rat and mouse models. Moreover, it was observed to inhibit NF-κB binding activity, a pivotal mechanism in shielding the liver from injury and cirrhosis triggered by hepatotoxins [167,168]. GL effectively protected against hepatocyte injury and cholestasis induced by α-naphthyl isothiocyanate (ANIT) by maintaining bile flow and ketoprofen glucuronide excretion [169]. In addition, it demonstrated protective effects against ANIT-induced liver injury by reversing bile acid metabolism disruptions and downregulating mRNA expressions of bile acid transport and metabolism proteins.

While evaluating the hepatoprotective effects of a GL product during alcohol consumption, normal volunteers were enrolled in a clinical study in which participants consumed vodka with either the GL product or a placebo. The alcohol-only group showed significant increases in liver enzymes (AST, ALT, GGT), while the GL group did not, suggesting that GL may support liver health during alcohol consumption [170]. In another clinical trial involving 50 healthy subjects to assess the effects of a GL/D-mannitol product on alcohol-induced oxidative stress and tissue damage, the product significantly reduced markers of oxidative stress and tissue damage after acute alcohol consumption [171].

GL mitigated pyrrolizidine alkaloid (PA)-induced hepatotoxicity in rats when administered over three consecutive days before exposure, significantly reducing serum transaminase levels and preventing extensive hepatocellular damage [172,173,174]. Moreover, GL showed efficacy against diazinon-induced toxicity, preserving liver and kidney function, ameliorating oxidative stress markers, and normalizing hematological and biochemical parameters. In cases of total parenteral nutrition (TPN)-associated liver injury, GL pretreatment dose-dependently reduced liver enzyme levels and histopathological damage by suppressing endoplasmic reticulum and reactive nitrogen stress factors [175,176]. In terms of detoxification and hepatoprotection, it was found to mitigate aflatoxin B1-induced liver toxicity. Abdel-Fattah et al. [177] showed that dietary supplementation with GL (500 ppm/kg) in rabbits improved liver enzyme levels and antioxidant markers, indicating its protective effect against mycotoxin-induced oxidative stress and organ damage.

2.5.4. Effect on Acetaminophen (APAP)-Induced Liver Injury

Combination therapy of GL with matrine (Mat) showed enhanced liver protection and anti-hepatocarcinogenic effects compared to either compound alone, reducing acetaminophen-induced hepatotoxicity and immunosuppression [178]. GL’s protective effect against acetaminophen-induced liver damage involves reversing fatty acid metabolism and bile acid alterations [179,180]. GL demonstrated therapeutic efficacy in acetaminophen-induced hepatotoxicity. Post-treatment with 40–160 mg/kg GL significantly attenuated mitochondrial damage and hepatocellular necrosis in mice by inhibiting nNOS expression and reducing tyrosine nitration, without interfering with APAP metabolism [181]. In mice, GL rapidly attenuated APAP-induced liver injury by inhibiting TNFα-induced hepatocyte apoptosis [182].

2.5.5. Effect on LPS/D-Galactosamine and HMGB1-Mediated Liver Injury

Studies on LPS and D-galactosamine (GalN)-induced liver injury in mice revealed that MMP-9 expression increased significantly in liver tissues 6–8 h after LPS/GalN treatment. Pretreatment with GL (50 mg/kg) or an MMP inhibitor (5 mg/kg) suppressed increases in ALT and AST levels. GL also inhibited MMP-9 mRNA and protein expression and reduced the infiltration of inflammatory cells expressing MMP-9 [183]. In acute hepatitis induced by LPS and D-galactosamine, GL (ED50: 14.3 mg/kg) reduced ALT levels and modulated inflammatory responses, including neutrophil and macrophage infiltration. While GL did not affect TNF-α, IL-6, IL-10, and IL-12 production, it significantly inhibited IL-18 production and responsiveness [184]. Using an LPS-induced mouse model of acute liver injury (ALI) and LPS-stimulated liver macrophages, GL was shown to significantly reduce inflammation by targeting and inhibiting HMGB1, a key pro-inflammatory mediator. Additionally, GL suppressed the PI3K/mTOR signaling pathway, further attenuating macrophage activation, inflammatory cytokine release, and apoptosis [185]. GL also blocked the extracellular release of HMGB1 in LPS/GalN-triggered liver injury, reducing serum ALT, AST, and HMGB1 levels and preventing apoptosis of hepatocytes [186]. Furthermore, GL inhibited apoptosis in LPS/D-galactosamine-induced hepatic injury by preventing the binding of HMGB1 protein to the Gsto1 promoter region [187]. In the case of HMGB1-induced liver damage, it effectively inhibited hepatocyte apoptotic process in Huh BAT cells promoted by HMGB1 through a p38-dependent mitochondrial pathway. It also prevented HMGB1-induced cytochrome c release and p38 activation in Huh-BAT cells [188].

2.5.6. Effect on Ischemia–Reperfusion Injury

GL’s role in I/R injury was also significant. In models of hepatic I/R, GL reduced serum liver enzyme levels, lipid peroxides, and apoptosis, while preserving glutathione content. It also attenuated cold ischemic injury in liver storage and reduced I/R-induced microcirculatory changes, supporting its hepatoprotective effects through HMGB1 signaling pathway modulation [189,190,191,192,193,194]. Similarly, in a myocardial I/R injury model using rats (in vivo) and H9C2 cardiomyocytes under hypoxia/reoxygenation (H/R) conditions (in vitro), GL improved cardiac function by reducing inflammation (HMGB1, TNF-α, IL-6, IL-18, IL-1β), oxidative stress (by lowering MDA and increasing GSH, CAT, and SODs), and ferroptosis (by decreasing TUNEL-positive cells and modulating ferroptosis-related gene and protein expression). GL’s efficacy was comparable to that of TAK-242, a TLR4-specific inhibitor. Mechanistically, GL exerted its effects via inhibition of HMGB1-TLR4 signaling and restoration of GPX4 [195].

A study by Zheng and Lou [196] investigates the protective effects of GL in immune-mediated liver injury using primary cultured rat hepatocytes. The results show that GL reduces inflammation, oxidative stress, and cell death, offering hepatoprotective benefits. It significantly protects against hepatic I/R injury in rats by attenuating liver damage, inflammation, oxidative stress, autophagy, and apoptosis. GL, when administered intraperitoneally at doses of 100 mg/kg and 200 mg/kg, showed hepatoprotective effects through activation of the Nrf2/HO-1 signaling pathway, with the higher dose showing greater efficacy [197].

2.5.7. Effect on Nonalcoholic Fatty Liver Disease and NASH

In nonalcoholic steatohepatitis (NASH) models, GL and its metabolite glycyrrhetinic acid improved hepatic steatosis, inflammation, and fibrosis by modulating bile acid homeostasis and inhibiting inflammasome activation. These findings suggest GL as a potential therapeutic option for NASH [198]. It inhibited mitochondrial permeability transition, oxidative stress, and cytochrome c release. Furthermore, GL improved nonalcoholic steatosis by modulating biochemical parameters and downregulating UCP2 gene expression [199].

2.5.8. Effect on Ferroptosis

Wang et al. [200] investigated the anti-ferroptosis effects of GL in acute liver failure (ALF) in which ALF was induced in L02 hepatocytes and mice using TNF-α, LPS, and D-galactosamine. Results showed that GL treatment reduced liver damage by lowering HMGB1 expression, a key driver of ferroptosis, and increasing protective proteins like Nrf2, HO-1, and GPX4. Additionally, GL decreased oxidative stress markers (LDH, ROS, MDA, Fe²⁺) and increased antioxidant levels (GSH).

2.5.9. Effect on Bile Acid Regulation and PXR Activation

When examined for its effects on pregnane X receptor (PXR)-mediated CYP3A expression and hepatoprotective activity, the treatment significantly increased CYP3A4 mRNA and protein levels in HepG2 cells through PXR-dependent transcriptional activation, as demonstrated by transient transfection experiments. This activation was confirmed using electrophoretic mobility shift assays (EMSA) [201]. To assess GL’s potential to prevent cholestasis-induced hepatotoxicity, mice were pretreated with GL before inducing intrahepatic cholestasis using lithocholic acid (LCA). GL, similar to the PXR activator pregnenolone 16α-carbontrile (PCN), mitigated increases in plasma ALT and AST activity, multifocal necrosis, and serum LCA levels. This hepatoprotection was linked to the induction of CYP3A11 (the mouse equivalent of human CYP3A4) and the inhibition of CYP7A1, facilitated by increased small heterodimer partner (SHP) expression, preventing toxic bile acid accumulation in the liver [202].

2.5.10. Effect on Viral Hepatitis and Related Conditions

GL has potential as an inhibitor of several viruses, including human immunodeficiency virus, varicella-zoster virus, cytomegalovirus, and hepatitis viruses, by modulating the immune response or inhibiting viral replication. It was found to enhance liver function recovery and regulated immune function in children with infectious mononucleosis complicated by liver impairment. Children treated with GL injection showed marked improvements in liver enzymes (ALT, AST), total bilirubin, and T lymphocyte subsets (CD3⁺, CD4⁺, CD8⁺ lymphocytes, and CD4⁺/CD8⁺ ratios) [203]. GL therapy was also found to be effective for liver dysfunction associated with cytomegalovirus (CMV) infection in immunocompetent children. Its therapeutic effect for liver dysfunction associated with CMV infection in immunocompetent individuals showed that liver dysfunction in four cases improved, and CMV disappeared from urinary samples after administration of intravenous GL by the age of 12 months [204,205]. Moreover, intravenous administration of the same formulation normalized liver function in infants with CMV infection and abnormal liver function or hepatomegaly [203,206].

GL was found to be effective in managing non-A, non-B hepatitis, inhibiting hepatitis A virus replication [207], improving liver function tests in patients with chronic hepatitis [208], and managing patients with both acute and chronic viral hepatitis [209]. In vitro, GL suppressed HBsAg secretion, causing its accumulation in the Golgi apparatus and inhibiting sialylation, indicating a unique mechanism by which GL affects HBsAg processing and secretion [210]. In HepG2.2.15 cells, GL variably affected HBeAg and HBV DNA levels at specific concentrations. It upregulated TLR2 and TLR4 expression and showed a negative correlation between cell proliferation and HBeAg/HBV DNA levels [211]. Active metabolites of GL were studied in guinea pigs, revealing glycyrrhetic acid as the most active form. GL at therapeutic concentrations suppressed HBsAg secretion [212].

2.5.11. Effect on Chronic Hepatitis B

It also improved serum transaminase levels in a chronic hepatitis B patient experiencing an acute exacerbation [213]. A clinical study involving 17 patients with chronic hepatitis B treated with GL followed by human lymphoblastoid interferon found that 11 patients lost DNA polymerase (DNA-p) activity, and 10 became HBeAg-negative. The therapy appeared effective in patients with higher ALT levels and lower initial DNA-p activity, which suggested that GL may prime the immune system for subsequent INF treatment [214]. In a combination therapy for chronic hepatitis B, ten chronic hepatitis B carriers received GL withdrawal and continuous human fibroblast interferon treatment. After 36 weeks, three patients became HBeAg-negative, with one showing undetectable DNA-p. Transaminase levels decreased in nine patients, and GL exhibited antiviral and corticoid-like effects in four and three patients, respectively. Interferon significantly reduced DNA-p and increased transaminase levels, with side effects primarily seen in patients receiving interferon [215]. A case study of a chronic HBV carrier with NHL, who developed HBV hepatitis following chemotherapy, showed that treatment with lamivudine and GL successfully controlled HBV replication and normalized transaminase levels [216].

2.5.12. Effect on Chronic Hepatitis C

Clinically, GL enhanced the effectiveness of interferon therapy in patients with interferon-resistant chronic hepatitis C [217] and showed an enhancing effect in combination with ribavirin in renal allograft recipients suffering from chronic hepatitis C [218]. In another clinical study, eight patients resistant to interferon therapy were treated with IFN combined with SNMC. This combination led to a 70% decrease in ALT levels and a reduction in serum HCV RNA in some patients, though overall benefits were not significantly greater than IFN alone [219]. Similarly, a study on 28 patients with chronic active hepatitis (CAH 2B) found that combining IFN with SNMC improved ALT normalization and HCV RNA clearance more than IFN alone, though not significantly. Histological improvement was more frequent with the combination therapy [220]. It demonstrated a reduction in HCV titers and showed a synergistic effect with INF-α [221]. In a study comparing INF-α-2b + ribavirin to INF-α-2b + GL for chronic hepatitis C, the former showed better responses but higher rates of leukopenia and anemia [222]. An evaluation of the efficacy and side effects of IFN-β therapy with or without GL (SNMC) in 14 patients with chronic active hepatitis C showed no significant differences in therapeutic efficacy or side effects between the two groups. However, γ-glutamyl transpeptidase levels were higher in the group treated with SNMC. A further study by Itoh et al. [223] on 27 chronic hepatitis C patients unresponsive to interferon therapy found that GL (60 mL of SNMC, thrice weekly for 16 weeks) reduced serum aminotransferase levels by half in 74.1% of patients, though 25.9% showed no improvement.

Long-term treatment with SNMC in chronic hepatitis C patients was found to reduce the development of liver cirrhosis and hepatocellular carcinoma (HCC). A multicenter study reported significant improvements in ALT levels and better liver histology with SNMC treatment [224]. A retrospective study assessed the long-term impact of SNMC on preventing HCC in patients with chronic hepatitis C. Among 453 patients, 84 treated with SNMC exhibited significantly lower 10- and 15-year cumulative HCC incidence rates compared to 109 patients who received other treatments. The relative risk of HCC was reduced by 2.49 times in the SNMC group [225]. A cohort study by Ikeda et al. [226] also found that GL significantly reduced the incidence of HCC in interferon-resistant chronic hepatitis C patients.

Clinical studies confirm that GL effectively reduces ALT levels in chronic hepatitis C patients but does not significantly affect HCV-RNA levels. In a European study of 57 patients, GL administered thrice weekly for 4 weeks resulted in a 26% decrease in ALT levels compared to a 6% decrease in the placebo group, with no major side effects [227]. Another study of 44 patients assessed pseudo-aldosteronism with varying doses of GL and found minor, reversible increases in blood pressure and decreases in aldosterone levels in the highest dosage group [228]. Additionally, a study on short-term effects in European patients showed that GL administered three or six times per week for 4 weeks significantly decreased ALT levels (26% and 47%, respectively), with the effect disappearing after treatment cessation. The six-times-weekly regimen was more effective than the thrice-weekly regimen [229]. A 26-week trial in chronic hepatitis C patients demonstrated that maintaining GL treatment three or six times weekly could sustain ALT responses. However, no significant histological improvements were noted after six months [230]. A phase III trial investigated GL’s efficacy in chronic hepatitis C non-responders to interferon + ribavirin. Administered intravenously 5×/week or 3×/week, GL significantly reduced ALT levels compared to placebo after 12 weeks. Improvement in necro-inflammation and fibrosis was observed after 52 weeks, with good tolerability [231]. A pilot study of a GL suppository showed it to be as effective as intravenous administration in reducing ALT levels, with no serious side effects [232]. In patients receiving GL, small intermittent phlebotomies before injection significantly reduced ferritin and ALT levels, suggesting an effective combined treatment for chronic hepatitis C [233].

While investigating the in vitro effects of GL on serum enzymes in chronic hepatitis patients, Akagi et al. [234] found that at a concentration of 40 μg/mL, it inhibited transaminase activity. It also inhibited the activities of serum LDH and LAP whereas activated acid RNase. Combining oral lamivudine with intravenous GL for subacute hepatitis was found to be a safe and effective treatment for subacute hepatitis caused by Hepatitis B and E viruses [235]. Fujisawa et al. [236] explored potential of GL in treating chronic viral hepatitis and inhibiting the complement system. It was found to block the cytolytic (cell-destroying) activity of complement by inhibiting the formation of the membrane attack complex without impacting immune adherence.

2.5.13. Effect on Autoimmune, Drug-Induced, and Acute Hepatitis

In patients with acute sporadic hepatitis E, intravenous GL led to significant clinical and biochemical improvement, reducing total bilirubin and normalizing liver enzyme levels without reported side effects [237]. In Japan, GL is used for drug-induced hepatitis, although rare complications like SNMC-induced allergic hepatitis have been reported. Its efficacy for anti-tuberculosis drug-induced hepatitis remains inconclusive [238,239]. It modulates liver inflammation by enhancing IL-10 production by liver dendritic cells and has been effective in reducing ALT levels in acute autoimmune hepatitis, potentially preventing disease progression [240,241].

In a Concanavalin A (Con A)-induced hepatitis mouse model, it reduced liver injury by decreasing inflammatory cytokines (IFN-γ, IL-6, IL-17) and serum ALT levels together with lowering the inflammatory molecule HMGB1 and increased IL-25 production, promoting a rise in protective Gr-1 + CD11b + MDSCs, which help resolve immune reactions [242]. Similarly, it protected against anti-Fas antibody-induced hepatitis in mice by inhibiting plasma aminotransferase activity and acting upstream of CPP32-like protease activation [243]. A mechanistic representation of its effect in reducing liver inflammation and fibrosis through immune modulation, via inhibition of CD4⁺ T cell proliferation and Th1/Th2/Th17 cell activation, and enhancement of antifibrotic cytokines IFN-γ and IL-10, is shown in Figure 3.

2.5.14. Effect Against Hepatocellular Damage in Metabolic Syndrome

GL has shown promise in treating metabolic syndrome complications, including insulin resistance, hyperglycemia, dyslipidemia, and oxidative stress. In a study where metabolic syndrome was induced in rats via a fructose-rich diet, GL (50 mg/kg, i.p.) reduced blood glucose, insulin, and lipid levels. It also decreased oxidative stress markers and improved antioxidant enzyme activity, restoring levels to near normal. Additionally, it increased PPARγ and GLUT4 protein levels in skeletal muscle, enhancing fatty acid oxidation and glucose homeostasis [244]. Further research demonstrated GL’s potential against metabolic syndrome-induced liver damage. It reduced oxidative stress, hepatic inflammation, and apoptosis in fructose-fed rats [245]. GL also improved liver mitochondrial function, reducing oxidative stress and enhancing electron transport chain activity in metabolic syndrome [246].

2.6. Effect of Glycyrrhizin on Urinary System

2.6.1. Effect on Acute Kidney Injury and Nephrotoxicity

In a study on early-phase ischaemia–reperfusion induced acute renal failure in rats, GL administration (200 mg/kg/day) significantly improved renal function and reduced structural damage in renal tissues. GL also restored urinary flow rate, solute-free water reabsorption, and creatinine clearance [247]. Sohn et al. [248] investigated its effects on gentamicin-induced acute renal failure in rats. Their findings demonstrated that GL restored the expression of aquaporin 2 water channels, reduced urine output, and partially normalized renal function parameters. Cadmium-induced renal dysfunction, often considered irreversible, was treated in rabbits with intravenous GL (SNMC). Despite high renal cadmium levels, this treatment led to improvements in hepatic injury, reduced plasma cadmium, and enhanced renal function [249].

2.6.2. Effect on Kidney Inflammation

It also exhibited a protective effect in a Con A-induced nephritis model by upregulating IL-25 expression in renal tissues, which subsequently promoted the polarization of macrophages toward the anti-inflammatory M2 phenotype. This polarization enhanced IL-10 secretion and reduced IL-1β production, thereby mitigating kidney inflammation. Mechanistically, IL-25 downregulated TLR4 expression on macrophages, and TLR4 was identified as essential for IL-25-mediated immunoregulation. These findings suggest that GL alleviates kidney injury through modulation of the IL-25/TLR4/M2 macrophage axis [250].

2.6.3. Effect on Adriamycin-Induced Nephropathy and Glomerulosclerosis

GL also showed protective effects on adriamycin-induced nephropathy in rats. It reduced urine protein levels, improved renal function, and alleviated renal morphological changes. Additionally, GL decreased the expression of laminin in renal tissues, indicating its potential in slowing the progression of glomerulosclerosis [251]. In nephrotic syndrome induced by adriamycin, GL significantly improved renal function, reduced proteinuria, and decreased expression of fibrotic markers [252]. In early glomerulosclerosis, GL reduced urine protein, improved renal function, and lowered the expression of TGF-β1, CTGF, and TIMP-1. These histopathological improvements suggest GL’s potential to mitigate the severity of glomerulosclerosis in rats [253].

2.6.4. Effect on Hemodialysis-Associated Oxidative Stress

Takeshita et al. [254] explored the preventive effects of GL on platelet-neutrophil aggregation and ROS production during hemodialysis (HD). In vitro experiments were conducted with heparinized blood exposed to polysulfone (PS) HD membranes. The results indicated that GL inhibited microaggregate formation and ROS production but did not affect P-selectin expression. Despite this, it still prevented platelet-leukocyte aggregation and neutrophil activation. Based on the findings, GL may help prevent HD-related inflammation and oxidative stress.

2.6.5. Effect on Renal Disorders

The combined treatment of GL and lamivudine for hepatitis B virus-associated glomerulonephritis in 80 patients showed significant improvements in urinary protein and serum albumin levels in the treatment group compared to the control group, with fewer adverse effects [255]. Additionally, GL, in combination with antiplasmin, was found effective in treating idiopathic renal hematuria [256].

2.7. Effect of Glycyrrhizin on Endocrine System

2.7.1. Effect on Pancreas and Its Function

GL has demonstrated significant protective effects against pancreatic injury in various experimental models. It has significant potential in reducing pancreatic inflammation and fibrosis through various mechanisms. In taurocholate-induced acute pancreatitis in rats, GL treatment significantly lowered levels of amylase, lipase, AST, urea, IL-6, TNF-α, and MPO, decreased pancreatic tissue MPO activities and MDA levels, and reduced acinar cell necrosis, hemorrhage, and edema [257]. In cerulein-induced AP in mice, GL treatment reduced serum amylase and lipase activities, lowered MCP-1 and MIP-2 levels, and decreased inflammatory cell infiltration in pancreatic tissue [258]. Another study demonstrated that GL significantly reduced serum levels of amylase, TNF-α, IL-6, and HMGB1, improving pancreatic lesions in a dose-dependent manner [259]. Zhang et al. [260] identified MAPK3 as a primary target through which GL exerts its protective effects in treating acute pancreatitis. It formed stable interactions with MAPK3 and ameliorated pancreatic cell injury by inhibiting the MAPK/STAT3/AKT signaling pathway.

GL also protects against severe acute pancreatitis-associated cardiac injury (SACI) by inhibiting ferroptosis. In a rat model of severe acute pancreatitis induced by sodium taurocholate, GL treatment improved cardiac function, reduced histological damage, and lowered serum cardiac injury markers (CK-MB, cTnI). Mechanistically, GL restored the expression of Nrf2 and HO-1, key regulators in the Keap1/Nrf2/HO-1 antioxidant pathway [261]. In another study on traumatic pancreatitis in rats, GL administration after inducing pancreatic trauma significantly reduced serum levels of HMGB1, TNF-α, and IL-6, improved survival rates, and ameliorated pancreatic injury [262]. Further research showed GL protected the pancreas from DL-ethionine-induced damage, preserving normal pancreatic lobular structure and acinar cells [263]. In addition, GL showed protective effects, particularly in cadmium-storing cells.

GL inhibited pancreatic fibrosis in trinitrobenzenesulfonic acid (TNBS)-induced chronic pancreatitis, potentially by protecting pancreatic acinar cells from mast cell activation and inhibiting extracellular matrix synthesis by pancreatic stellate cells. In a study investigating the effects and mechanisms of GL on TNBS-induced pancreatic fibrosis, chronic pancreatitis was induced in male rats. GL (8 mg/kg, i.v.) significantly decreased the number of mast cells and the percentage of degranulation. Additionally, GL treatment resulted in a marked reduction in inflammation and fibrosis, with decreased expression of Collagen I and TGF-β1 [264]. In a chronic pancreatitis (CP) model induced by ethanol (36% for 4 weeks) and cerulein (20 µg/kg, i.p. for 3 weeks) in rats, oral GL (10 mg/kg/day, from week 3) significantly attenuated pancreatic damage. GL reduced ER stress by downregulating UPR genes (ATF4, GRP78, CHOP, XBP1), suppressed NF-κB-mediated inflammation, and ameliorated oxidative stress. It also preserved mitochondrial function by reducing calcium overload, restoring complex I activity, and lowering 4-HNE levels. These results suggest GL exerts pancreato-protective effects by targeting ER stress, NF-κB signaling, and mitochondrial dysfunction [265].

2.7.2. Mineralocorticoid Activity

GL’s mineralocorticoid activity is attributed to its inhibition of the enzyme 11β-dehydrogenase, which converts cortisol to cortisone. In a clinical study involving 18 healthy volunteers, administration of GL (225 mg/day) resulted in suppressed plasma renin activity, hypokalemia, and kaliuresis, effects similar to those of 9α-fluorocortisol. These effects were reduced when GL was co-administered with dexamethasone. Additionally, it increased urinary cortisol excretion and decreased cortisone levels [266].

2.7.3. Anti-Androgen Activity

When investigated for its effects on ovarian androgen production in rats, GL reduced testosterone production without altering delta 4-androstenedione and estradiol levels. It increased the estradiol/testosterone production ratio, indicating stimulated aromatase activity. GL inhibited testosterone synthesis and promoted estradiol synthesis by acting directly on the rat ovary [267]. GL (100 mg/kg, i.p.) was also found to improve glucose metabolism and ovarian function in a mouse model of polycystic ovary syndrome (PCOS) induced by dehydroepiandrosterone and high-fat diet. It reduced serum and ovarian HMGB1 levels, restored hormonal balance, improved insulin sensitivity and glucose tolerance, and normalized estrous cycles and ovarian morphology. Mechanistically, GL suppressed the TLR9/MyD88/NF-κB inflammatory pathway and activated the IR/p-Akt/GLUT4 insulin signaling pathway [268].

2.8. Effect of Glycyrrhizin on Skin and Skin-Related Disorders

GL has been used clinically for several years due to its beneficial effects on IgE-induced allergic diseases, alopecia areata, and psoriasis. Research has shown that GL exhibits anti-pruritic effects in clinical settings, notably for antihistamine-resistant pruritus in hemodialysis patients [269]. In animal models, it notably reduces scratching behavior induced by substance P, PAR-2 agonistic peptide, or leukotriene B4, but not by histamine. When administered intravenously at doses of 25 or 50 mg/kg in mice, it significantly inhibited passive cutaneous anaphylaxis. Additionally, it dose-dependently counteracted contractions in isolated rabbit ileum and guinea pig trachea induced by histamine, acetylcholine, and SRS-A, demonstrating its potential antihistaminic and antispasmodic effects [270]. When evaluating the efficacy and safety of GL (75 mg three times a day) combined with mizolastine (10 mg daily) in treating chronic idiopathic urticaria in 54 patients, the combination therapy showed superior results compared to mizolastine alone [271]. It was found that GL can effectively control sepsis from pseudomonal infections and restore antimicrobial peptide production in severely burned patients [272].

In a study investigating its impact on pseudomonal skin infections using a chimera model of thermal injury, GL-treated chimeras survived a lethal dose of P. aeruginosa infection, displaying human antimicrobial peptides at graft sites. Moreover, GL facilitated HBD-1 production in keratinocyte cultures with CD31⁺ IMC and eliminated HBD-1 inhibitors such as IL-10 and CCL2. Studies also support that GL can protect against UVB-induced damage in specific melanoma cells [273]. Its protective effect against UVB irradiation showed increased Bcl-2 expression in human melanoma (SKMEL-28) cells. Additionally, GL was reported to improve cutaneous and biochemical abnormalities in a patient with porphyria cutanea tarda [274]. Furthermore, GL has demonstrated protective effects against various other skin conditions, as detailed below.

2.8.1. Effect Against Psoriasis

Several studies have investigated the effects of GL in combination with other treatments for psoriasis. GL, especially when used with acitretin, offers significant clinical advantages in treating psoriasis, including enhanced efficacy, reduced inflammation, and better management of associated liver abnormalities. In a study involving 50 psoriasis patients, the combination of GL with acitretin was more effective than acitretin alone. Both treatment groups showed significant reductions in Th17 cell percentages and cytokine levels (IL-6, IL-17, IL-22, TGF-β) post-treatment, with more pronounced effects in the combined treatment group, resulting in higher clinical efficacy [275]. Additionally, GL reduced Th17 cell proportions and IL-22 levels in patients with psoriasis vulgaris, which correlated with its therapeutic efficacy [276].

For patients with generalized pustular psoriasis and liver test abnormalities, the combination therapy of acitretin (20–30 mg/day orally) and GL (150 mg/day orally) significantly improved psoriasis severity scores and liver enzyme levels in a study of nine patients. These patients maintained stable conditions over a 12-month follow-up without any side effects [277]. It has been found that GL ameliorates skin inflammation by interfering with key signaling pathways. It reduced ICAM-1 expression, inhibited monocyte adhesion to keratinocytes, and suppressed NF-κB/MAPK signaling pathways. GL also delayed the onset and reduced the severity of psoriasis-like inflammation in mice [278].

In an imiquimod-induced psoriasis mouse model, GL significantly improved psoriasis-like symptoms and skin pathology. It reduced serum IL-17A and IFN-γ levels. In vitro, 100 ng/mL IL-17A-stimulated HaCaT cells were used to mimic psoriatic keratinocyte activation. It inhibited the proliferation of these cells and suppressed the secretion of pro-inflammatory cytokines IL-6, TNF-α, and CCL20. Mechanistically, GL upregulated SIRT1, leading to inhibition of STAT3, phosphorylated STAT3, and acetylated STAT3 [279].

2.8.2. Effect Against Alopecia Areata

GL has been found effective against mild to severe alopecia areata, an autoimmune disorder that can lead to unpredictable hair loss. In a study involving 65 adult patients with mild to moderate active alopecia areata, oral tablets of 25 mg GL taken three times per day, along with topical 0.05% halometasone cream applied twice daily for 24 weeks, resulted in a higher complete regrowth rate and total efficacy compared to the control group. The recurrence rate during follow-up was 12.3% without severe adverse events [280]. Furthermore, the combination of GL with betamethasone was also found effective against severe active alopecia areata. In a study involving 100 patients, the combination of oral GL tablets and betamethasone injections every three weeks for 24 weeks showed a higher efficacy rate and a lower relapse rate at 36 weeks compared to betamethasone alone, with no severe adverse effects observed [281].

2.8.3. Effect Against Eczema and Keloids

In a study evaluating the effects of GL on a mouse model of 2,4-dinitrochlorobenzene-induced atopic dermatitis-like symptoms, the treatment inhibited the HMGB1 signaling cascade and ameliorated atopic dermatitis symptoms. It indicates that HMGB1 plays a role in the pathogenesis of atopic dermatitis. In vitro studies on P815 mouse mast cells demonstrated that GL inhibited HMGB1-induced mast cell activation by reducing Ca²⁺ influx, downregulating CD117, and suppressing NF-κB signaling. These findings suggest that GL could be a potential therapeutic agent for cutaneous inflammation [282]. A clinical study involving 199 patients with chronic eczema assessed the efficacy and safety of GL (75 mg orally, three times a day) combined with corticosteroid therapy (0.1% mometasone furoate, applied topically once a day). The combination therapy significantly improved symptoms, including greater reductions in EASI scores and higher proportions of patients achieving almost clear IGA scores. Additionally, pruritus was significantly reduced, and eczema recurrence was lower, with minimal adverse events reported [283].

Interestingly, GL shows promise as a keloid treatment by regulating the extracellular matrix and autophagy [284]. It suppressed fibroblast proliferation and ECM expression in keloids while reducing autophagy. Additionally, GL inhibited HMGB1, leading to downregulation of ERK1/2, Akt, and NF-κB, as well as decreased expressions of TGF-β, Smad2/3, and ERK1/2.

2.8.4. Effect Against Hyperpigmentation

The effects of GL on mouse melanoma (B16) cells, when investigated, showed inhibition of cell growth, caused morphological changes, and stimulated melanogenesis at high concentrations [285]. It increased tyrosinase mRNA, protein levels, enzyme activities, and melanin content, with no observed cytotoxicity [286]. GL-induced melanogenesis was linked to the transcriptional activation of tyrosinase-related protein-2 and involved the activation of activator protein-1 and cyclic AMP response element promoters, without affecting the NF-κB promoter. The process was mediated through cAMP signaling, as evidenced by the inhibition of GL-induced melanogenesis by a protein kinase A inhibitor (H-89), confirming GL’s action upstream of protein kinase A [287].

A 30-day study assessed the efficacy and safety of combining oral tranexamic acid (500 mg per day) and GL (150 mg per day) for treating recalcitrant Riehl’s melanosis in ten patients. This therapy led to moderate improvement in most patients, with significant reductions in melanin and erythema indices [288]. Another study combined salicylic acid chemical peels (30%), oral GL (150 mg/day), and vitamin C (100 mg/day) in three patients, resulting in significant improvement without any noticeable side effects [289]. These findings suggest that GL, when used in combination with other treatments, may effectively manage Riehl’s melanosis.

2.8.5. Effect Against Vitiligo

A clinical study involving 144 patients examined the use of GL, ultraviolet B light (UVB), or a combination of both to treat active-stage generalized vitiligo. The treatment significantly improved the vitiligo area scoring index of the affected body surface, resulting in repigmentation of lesions in all patients. The combination of GL and UVB was found to be more effective in improving the disease stage, with limited and transient adverse events [290]. In another study by Mou et al. [291] evaluating its protective effects against oxidative stress in human melanocytes, a major cause of vitiligo, human primary melanocytes exposed to H₂O₂ and treated with GL showed significantly improved cell viability, reduced apoptosis, and decreased reactive oxygen species levels. GL activated the Nrf2 pathway and induced the expression of heme oxygenase-1. Knockdown of Nrf2 or inhibition of heme oxygenase-1 reversed GL’s protective effects, demonstrating that GL protects melanocytes from oxidative damage via the Nrf2-dependent induction of heme oxygenase-1.

Its combination with fractional CO₂ laser and triamcinolone acetonide solution showed a better clinical effective rate in patients with vitiligo compared to treatment without GL. Additionally, IL-17 levels decreased, and TGF-β levels increased significantly, suggesting that when combined with fractional CO₂ laser and triamcinolone acetonide, GL can effectively treat vitiligo and modulate cytokine levels [292].

2.8.6. Effect Against Facial Rosacea and Acne Vulgaris

A clinical study by Li et al. [293] evaluated GL injection delivered via mesoderm therapy for treating facial rosacea in 58 patients. It was observed that GL-treated patients experienced significant improvements in erythema, flushing, telangiectasia, and papulopustules, along with reduced transepidermal water loss (TEWL) and increased stratum corneum hydration. Moreover, patients also showed a marked improvement in the dermatology life quality index when compared to the control group that received topical metronidazole-clindamycin liniment. Similarly, mesotherapy-based administration of GL injection in combination with clindamycin gel in 54 patients with moderate to severe acne. GL (40 mg) significantly improved clinical outcomes compared to clindamycin alone. GL-treated patients showed greater reduction in acne severity, enhanced skin barrier function as evidenced by decreased TEWL, and increased stratum corneum water content. Furthermore, this treatment approach effectively reduced inflammatory reactions and skin damage [294].

2.9. Effect of Glycyrrhizin on Ocular Diseases

A clinical study involving 37 patients evaluated GL eye drops (2.5%, twice daily for 28 days) for moderate dry eye disease. After 28 days, significant improvements were observed in tear film stability, symptoms, and overall ocular health [295]. In a rabbit model of ocular hypertension induced by triamcinolone acetonide (TA), GL decreased intraocular pressure and improved electrophysiological parameters such as flash ERG and VEP. It also mitigated TA-induced changes in ocular metabolism, particularly affecting sugar metabolism [296]. Research also highlighted GL’s protective effects against retinal damage caused by sodium iodate in mice. It protected the retinal pigment epithelium from sodium iodate-induced ROS and apoptosis by increasing Akt phosphorylation and the expression of Nrf-2 and HO-1. It also reduced retinal cell apoptosis, inhibited retinal thinning, decreased drusen, and improved retinal function [297].

In IGFBP-3 knockout mice, GL reduced neuronal and vascular damage in the retina through anti-inflammatory mechanisms [298]. It decreased ROS levels and the protein levels of HMGB1, TNFα, and IL-1β, showing effectiveness in reducing neuronal damage at two months and vascular damage at six months. For pathological corneal conditions, GL effectively inhibited corneal neovascularization (CNV) in an experimental model at 1%, with 3 drops thrice daily for 27 days [299]. It exerted potent anti-angiogenic and anti-inflammatory effects by targeting the HMGB1/NF-κB/miR-21 axis. In an alkali burn-induced CNV model, GL significantly reduced CNV and inflammation. GL suppressed HMGB1 expression (mRNA and protein), inhibited VEGF production via the HMGB1/NF-κB/HIF-1α pathway, and decreased pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) and chemokines (CCL2/CXCL5) and their receptors (CCR2/CXCR2). GL also improved corneal clarity by suppressing TGF-β1-induced myofibroblast differentiation, partially through downregulation of miR-21 [300]. It also demonstrated prophylactic effects against Pseudomonas aeruginosa keratitis in mice [301]. It protects against P. aeruginosa keratitis by downregulating key inflammatory mediators, particularly TLR4, TLR9, HMGB1, and RAGE, showing its potential as a therapeutic agent that modulates innate immune responses, with TLR9 playing a central role in its mechanism of action [302].

GL reduced HMGB1 levels, improved clinical outcomes, decreased inflammatory markers, neutrophil infiltration, and bacterial load in noncytotoxic P. aeruginosa infections. For cytotoxic strains, it improved outcomes but did not reduce HMGB1 levels. GL showed bacteriostatic activity against both strains, increased bacterial permeability, reduced biofilm formation, and decreased bacterial adherence [303]. Moreover, GL acted as a bioenhancer for tobramycin in treating Pseudomonas keratitis [304].

2.10. Effect of Glycyrrhizin on Metabolic Disorders

2.10.1. Effect on Obesity and Associated Complications

Previous studies have demonstrated the significant role of GL, either as a prophylactic measure or as a treatment, in combating insulin resistance associated with obesity. A study by Abo El-Magd et al. [305] on the anti-obesity effect of GL (50 mg/kg/day) on rats induced with a high-fat diet showed significant reductions in weight and insulin resistance. The treatment also normalized lipid profiles, decreased adipocyte size, reduced liver lipid deposition, decreased oxidative stress, and increased antioxidant capacity. Additionally, it lowered liver gluconeogenic enzyme mRNA levels and increased the expressions of insulin receptor, NrF2, and heme oxygenase-1 mRNA, along with promoting NrF2 nuclear translocation. A related study by Madhavadas and Subramanian [306], evaluating the cognitive-enhancing effect of a combination of Spirulina and GL in monosodium glutamate-induced obese aged rats, demonstrated that the combination of Spirulina (1 g/kg for 30 days) and GL (0.1 mg/kg on days 15 and 21) significantly reduced glucose, cholesterol, and leptin levels. Moreover, it improved cognitive functions and decreased hippocampal AChE activity, which suggested its potential in reversing cognitive dysfunctions associated with aging and obesity.

2.10.2. Effect on Diabetes and Associated Complications

While focusing on pancreatic repair and β-cell protection, it has been found that GL showed the reparative effects on streptozotocin-induced pancreatic injuries in rats. It promoted regeneration of lipid-storing cells containing lipid droplets and repaired pancreatic damage. In KK-Ay diabetic mice, long-term treatment with a 0.41% GL diet significantly suppressed postprandial blood glucose rise and improved oral glucose tolerance. This treatment also lowered blood insulin levels and prevented excessive water intake, without affecting food intake or body weight [307]. Similarly, in STZ-induced diabetic rats, GL (administered at doses ranging from 2.7 to 4.1 g/kg diet) alleviated key diabetic symptoms, including elevated blood glucose, glucose intolerance, and reduced insulin levels. It also reversed oxidative stress and reduced damage to the pancreas and kidneys, with effects comparable to the standard antidiabetic drug glibenclamide [308].

In Zucker diabetic fatty rats, GL administered over 4 weeks significantly reduced inflammation markers (HMGB1, TLR4, NF-κB) linked to DKD, while ameliorating glomerular injury. These findings suggest that GL may help mitigate the progression of DKD by inhibiting inflammation-related pathways [309].

GL effectively attenuates inflammation and oxidative stress in diabetic corneal pathology. It protected mouse corneal epithelial cells from high glucose-induced cytotoxicity and significantly reduced inflammatory and oxidative stress markers (e.g., HMGB1, IL-1β, TLRs, NLRP3, COX2, antioxidant enzymes) both in vitro and in diabetic mice. GL-treated diabetic mouse corneas showed suppressed expression of inflammatory genes and proteins. Ex vivo human diabetic corneas similarly showed elevated levels of these markers, supporting translational relevance [310].

In a STZ-induced diabetic rat model (8–12 weeks post-induction), GL treatment effectively attenuated myocardial remodeling. It reduced TNF-α expression, fibrosis, and collagen deposition, and significantly decreased apoptosis as shown by DAPI, BrdU, and caspase staining. The study confirmed modulation of relevant protein expression, which showed that GL mitigates hyperglycemia-induced cardiac damage by suppressing inflammation and apoptosis [311]. Moreover, in diabetic cardiomyopathy, GL restored connexin-43 expression and attenuated oxidative stress, cardiac atrophy, and fibrosis by inhibiting the TGF-β/p38MAPK and CXCR4/SDF1 signaling pathways while activating Nrf2 [312].

2.11. Effect of Glycyrrhizin on Gastrointestinal System

GL showed potential in modulating bacterial enzyme activity and immune responses, offering protective effects against atherosclerosis and radiation-induced sepsis. In a mouse model of gastrointestinal acute radiation syndrome, GL administration after exposure to 7 Gy γ-rays controlled mortality by reducing bacterial translocation and sepsis. This effect was achieved by modulating macrophage polarization, increasing Gas5 RNA, and decreasing miR-222 expression, which prevented the formation of inhibitory M2b macrophages and enhanced antibacterial defense [313]. The presence of GL stimulated the growth of Eubacterium sp. strain GLH, isolated from human feces, in a nutrient-poor medium by providing D-glucuronic acid. In mixed cultures, it enhanced the activity of β-glucuronidase and suppressed 3β-hydrosteroid dehydrogenase activity, leading to the conversion of GL to glycyrrhetic acid. However, glycyrrhetic acid inhibits bacterial growth, indicating a feedback mechanism that limits the proliferation of the bacteria. This suggests that while GL promotes bacterial growth initially, the resulting glycyrrhetic acid eventually suppresses it, maintaining a balance in bacterial population [314,315].

2.12. Effect on Different Cancers and Tumors

The potential of GL has been demonstrated in several studies, particularly in cancer prevention and immune system modulation. In vitro, GL (200 µg/mL) upregulated H-2 class I antigen expression and class I gene transcription in multiple tumor cell lines (L1210, L5178Y, P388, PI.HTR, NS-1, MethA, YAC-1, and EL-4) [316]. Additionally, in vivo studies revealed that GL administration (0.05–0.2 mL, i.p.) enhanced H-2Dd antigen expression in normal bone marrow, spleen, and peritoneal cells of BALB/c mice. These effects appeared to be independent of interferon production, suggesting a direct immunomodulatory mechanism that may enhance antitumor immunity [316]. GL’s role in enhancing cancer immunotherapy was further supported by Suzuki et al. [317], who administered the compound at 20 mg/kg to mice inoculated with Meth A tumor cells. While GL alone had weak antitumor effects (40% of mice showed no response, 45% experienced delayed tumor growth, and 15% exhibited tumor regression), the addition of adoptive transfer of allospecific cytotoxic T lymphocytes (CTLs) significantly boosted its efficacy. Similarly, Madhiba and Matsunaga [318] highlighted the potential for GL, in combination with TNF, lymphotoxin, and diethyldithiocarbamate, to enhance antiproliferative effects against Meth A tumor cells.

In studies exploring anticancer effects in hematologic and metastatic models, Malagoli et al. [319] found that GL (at doses of 25 µM and higher) inhibited the growth of human leukemia and lymphoma cell lines, showing a particularly strong effect against leukemia cells and outperforming equimolecular doses of daunorubicin. Kobayashi et al. [320] investigated GL at 10 mg/kg in mice inoculated with B16 melanoma cells and observed a dramatic reduction in pulmonary metastasis, with the GL-treated group having only 48 metastatic colonies compared to 208 in the control group. Additionally, GL-treated CD4⁺ T cells inhibited metastasis by 84%, underscoring the compound’s immunomodulatory effects.

In relation to mechanistic modulation of cancer-related signaling pathways, Hsiang et al. [321] reported that GL (10–50 µM) induced the AP-1 transcription factor in both untreated and tumor promoter-treated cells. While it stimulated AP-1 activity in untreated cells, it inhibited TPA-induced AP-1 activation in treated cells, suggesting a context-dependent dual regulatory role. GL was also shown to target HMGB1-mediated tumor inflammation. Smolarczyk et al. [322] demonstrated that GL inhibited HMGB1, a pro-inflammatory protein released during tumor cell necrosis. This treatment reduced cell proliferation, migration, angiogenesis, and inflammation in tumor tissues, enhancing the anticancer effects of the CAMEL peptide, which induces necrotic cell death in tumors. These findings suggest GL’s ability to modulate HMGB1-related inflammation and tumor regrowth could be a valuable strategy for improving cancer therapies. Regarding chemoprevention and combination strategies, Yasukawa et al. [323] explored the combined use of GL (20 mg/kg) and caffeine in a two-stage carcinogenesis model. The combination proved more effective than either compound alone in inhibiting tumor promotion by TPA in mice. The study demonstrated that the combination significantly reduced both tumor incidence and size, with the combined treatment showing a more pronounced effect.

A comprehensive summary of the cancer types, experimental models, doses of GL, the mechanisms of action, and the results obtained from the studies, along with the references for each study, is provided in

Table 1. Effect of glycyrrhizin against different cancers.

Cancer Type	Model	Dose	Results	Mechanism	Reference
Skin Cancer	Sencar mice (in vivo)	Oral feeding of 100–250 mg/kg	Prolonged tumor latency, reduced tumor number, and inhibited [3H]DMBA–DNA binding.	Inhibits carcinogen metabolism and DNA adduct formation	[324]
	Swiss albino mice (in vivo)	2.0 and 4.0 mg/0.2 mL acetone/animal (topical)	Decreased lipid peroxidation, ODC activity, and DNA synthesis while restoring GSH and its enzymes, protecting against TPA-induced oxidative stress	Reduces oxidative stress, lipid peroxidation, and inflammation; restores GSH, lowers ODC	[325]
Lung Cancer	A549 and NCI-H23 lung adenocarcinoma cell lines, and female nude mice for tumor formation (in vivo/in vitro)	0.25–2.5 mM	Induced apoptosis via TxAS inhibition	Induces apoptosis; inhibits TxAS, STAT3, Survivin, HMGB1, JAK/STAT	[326]
	Nude mice with human lung adenocarcinoma xenografts (in vivo)	GL (15, 45 and 135 mg/kg), cisplatin (2.5 mg/kg)	Co-treatment reduced TxAS and PCNA expression, improved liver/kidney function, and suppressed tumor growth via the TxA2 pathway.	Suppresses PCNA, TxA2 signaling	[327]
	Human NSCLC PDX mice model (in vivo)	100 mg/kg, i.p.	Inhibited migration and invasion of lung cancer cells by suppressing HMGB1 levels; reduced JAK/STAT pathway activity.	Inhibits angiogenesis via HMGB1/RAGE, TLR4, NF-κB	[328]
	HCC827 and A549 non-small cell lung cancer (NSCLC) cells (in vitro)	3 mmol/L	Upregulated miR-142, leading to downregulation of ZEB1 and inhibition of malignant behaviors in NSCLC cells.	Regulates miR-142/ZEB1 axis	[329]
	B16 melanoma metastasis in mice (in vivo)	15 mg/kg orally, once every 2 days for 2 weeks	Reduced melanoma cell extravasation into lungs.	Modulates HMGB1/RAGE and TLR4 signaling; suppressed EMT and angiogenesis pathways.	[330]
	A549 cells (in vitro)	GL (0.25–8 mM); cisplatin (10–160 µM)	Reduced A549 cell viability; combined treatment reduced cell colony-forming ability, induced apoptosis and arrested the cell cycle at the G2 phase	Increases pro-apoptotic and DNA damage markers (Bax, cleaved-caspase-3, γH2AX, p-Chk1, p-p53); reduces anti-apoptotic and cell cycle proteins (Bcl-2, cyclin D1, CDK2, CDK4).	[331]
Liver Cancer/Hepatocellular Carcinoma (HCC)	Diethylnitrosamine-treated BALB/c mice (in vivo)	2 mg/kg, 3×/week for 2 weeks	Reduced the number of liver tumors and HCC incidence significantly compared to control. Liver function improved (AST, albumin)	Inhibits tumor formation and HCC development	[332]
	Sprague-Dawley rats (in vivo)	240–480 mg/kg	Induced liver enzyme activity; concern for cotoxicity and cocarcinogenic effects	Induces cytochrome P450 enzymes (CYP3A, CYP1A2, CYP2B1) in liver	[333]
	HepG2 (in vitro)	1.0 mmol of GL for 48 h	Decreased the expression of TIMP-1, indicating anti-fibrotic and potential anticancer effects	Down-regulates TIMP-1 gene expression via inhibition of its promoter	[334]
	Rats treated with lead acetate (100 mg/kg, i.p.) (in vivo)	150–300 mg/kg GL (oral)	Mitigated lead-induced oxidative stress, liver damage, and tumor promotion	Inhibits ODC activity, and DNA synthesis in liver	[335]
	Cisplatin-resistant Huh7 HCC cell line (in vitro)	GL (5 and 100 μg/mL), co-treatment with cisplatin (5 μg/mL)	Co-treatment reversed resistance and decreased cell viability, improving cisplatin retention in resistant cells	Modulates MRPs (MRP2, MRP3, MRP4, MRP5) and enhances cisplatin accumulation	[336]
	HepG2 (in vitro)	GL used in the form of Monoammonium GLate (10 mg/60 mm dish)	Increased junB gene expression, suggesting its potential as a tumor suppressor	Inhibits c-jun oncogene expression, stimulates junB gene	[337]
	40 liver carcinoma patients treated with GL capsules (clinical)	1 capsule/day (dose size not available)	Prevented elevated Asn levels in liver cancer patients, inhibited clopidogrel metabolism, and demonstrated mild toxicity	Normalizes Asn levels and alters clopidogrel metabolism	[338]
	HepG2 and MHCC97-H cells, xenograft mouse model	(1 and 2 mmol/L)	Induced autophagy and cytotoxicity, inhibited tumor growth in xenograft models	Induces autophagy, inhibits Akt/mTOR, and activates ERK1/2 signaling pathways	[339]
	HCC, HUVECs (in vitro)	<100 μM	Reduced cell proliferation, migration, and angiogenesis; decreased levels of p-STAT3, Survivin, LC3-I, P62, and VEGF-A	Inhibits of STAT3/Survivin pathway and induces autophagy; increases LC3-II and Beclin1, and reduces LC3-I, P62, and VEGF-A	[340]
	H22 tumor-bearing mice, RAW 264.7 macrophages (in vivo/in vitro)	DOX/GL-ALG nanogel particles (5.25, 21, 84 μg/mL), in vitro and 2.5 mg/kg, in vivo	Increased DOX bioavailability, reduced macrophage activation, and enhanced anticancer efficacy against HCC in vivo	Enhances targeting, reduces macrophage phagocytosis, and regulates apoptosis pathways	[341]
Endometrial Cancer	Mice (Estradiol-induced carcinogenesis) (in vivo)	Glycyrrhizae radix and GL (0.0625%)	Decreased COX-2, IL-1α, and TNF-α; reduced adenocarcinoma incidence	Suppresses COX-2, IL-1α, TNF-α gene expression	[342]
Breast Cancer	MDA-MB-231 TNBC cells (in vitro)	GL (5–80 μM/L), Glycyrrhetinic acid (GA)	Induced apoptosis and enhanced etoposide-induced cytotoxicity	enhances TOPO 2A expression, sensitizes cells to etoposide, inducing apoptosis via MAPK and AKT pathways	[343]
	MDA-MB-231 and BT549 cells (in vitro)	5–40 μM	Decreased migration and invasion	Upregulates miR-200c and E-cadherin expression	[344]
Gastric Cancer	KATO III, HL-60 (in vitro)	Not available	Growth inhibition observed, apoptosis induced, caspase involvement confirmed	Induces apoptosis via caspase activation and DNA fragmentation	[345]
	BGC-823 (in vitro)	40 μM/L	Decreased proliferation, migration, and adhesion; reduced expression of β-catenin, Bcl-2, CyclinD1, and survivin.	Modulates Wnt/β-catenin signaling, inhibits cell adhesion and migration	[346]
Prostate Cancer	LNCaP, DU-145 (in vitro)	Not available	Inhibition of cell proliferation, induction of apoptosis	Induces apoptosis via caspase-independent pathways; causes DNA damage	[347]
	DU145 (in vitro)	25–200 μM	Reduced EMT markers, blocked migration and invasion.	Inhibits HMGB1-induced EMT	[348]
Cervical Cancer	HeLa (in vitro)	20–640 μM	Reduced cell viability, DNA fragmentation, ROS increase, apoptosis induction, and G0/G1 cell cycle arrest.	Induces ROS-dependent apoptosis, mitochondrial depolarization, and cell cycle arrest at G0/G1 phase	[349]
	C33A (in vitro)	10–100 μM	Induced apoptosis and G0/G1 cell cycle arrest.	Activates caspase, downregulates notch signaling, upregulates p21	[350]
	End1/E6E7, C33a (in vitro)	50 μmol/L	Decreased proliferation, migration, and invasion of C33a cells and increased apoptosis.	Modulates protein kinase B/glycogen synthase kinase-3β/β-catenin signaling pathway	[351]
Colorectal Cancer	SW48, CCD-18Co (in vitro); azoxymethane/dextran sodium sulfate-induced mice (in vivo)	12 μM/15 mg/kg/day orally (mice)	Inhibition of growth, apoptosis, autophagy, and reduced migration/invasion; inhibits the inflammatory response; inhibits DNA damage and cancer stem cell proliferation and dedifferentiation.	Targets MMP-9 and MMP-2 expression; inhibits HMGB1-TLR4-NF-κB signaling	[352,353]
Bone Cancer (Osteosarcoma)	U-O2S (in vitro)	10–40 μM	Sensitized cells to doxorubicin, reversing drug resistance under hypoxia.	Antagonizes hypoxia-induced chemoresistance, inhibits HMGB1 signaling	[354]
Submandibular Gland Cancer	Mice submandibular gland fibrosarcoma cell line (in vitro)	0.6 mg/mL and above	Inhibited cell proliferation above 600 mg/L.	Blocks G1 to S phase transition in the cell cycle	[355]

.

2.13. Effect of Glycyrrhizin on Immune System

GL was found to be a potent immunomodulator with diverse effects across both innate and adaptive immunity. It modulates T cell apoptosis, dendritic cell maturation, macrophage cytokine signaling, and mast cell stabilization, making it a promising agent in managing inflammatory and immunocompromised conditions.

2.13.1. Effect on T-Cell Regulation, Apoptosis, and IL-2 Signaling

GL exerts selective immunomodulatory effects on T lymphocytes by inducing dose-dependent apoptosis, especially in CD4⁺ and CD8⁺ T cells, while sparing B cells. In a murine splenocyte and thymocyte model, GL concentrations ranging from 0.1 to 1.0 mM led to DNA fragmentation and apoptosis, primarily through mitochondrial membrane potential (ΔΨm) reduction [356]. Regarding cytokine regulation, at concentrations of 0.1–0.5 mM, GL modulates interleukin-2 (IL-2) expression differentially in immature and mature T cells. In mature T cells, it enhances IL-2 signaling by upregulating IL-2 receptor expression through late-stage T-cell receptor signaling, bypassing early phosphorylation events and c-fos activation [357]. Conversely, in immature thymocytes, GL increases IL-2 secretion while suppressing cell proliferation, thereby uncoupling the IL-2 response from cell division [358]. This dual immunoregulatory behavior, dependent on cell maturity and external stimuli, underscores GL’s potential in modulating T cell responses under both stimulatory and suppressive immune conditions [359].

2.13.2. Effect on Macrophage Activation and Innate Immune Modulation

GL primes macrophages to enhance IL-12 production, both the p70 heterodimer and p40 subunit, in response to LPS stimulation. This effect is mediated via NF-κB activation and occurs independently of IFN-γ or GM-CSF, indicating a direct modulatory action on macrophage signaling pathways [360]. In this study, murine peritoneal macrophages were pretreated with GL at 200 μg/mL for 2 h before LPS stimulation. In a thermal injury mouse model, where IL-12 expression is typically suppressed, GL administered intraperitoneally at 10 mg/kg/day for 5 days restored IL-12 levels, suppressed Th2-skewed immune responses, and promoted regulatory CD4⁺ T cell populations, thus correcting the burn-induced Th2 bias [361]. Additionally, GL modulates intracellular second messengers such as cyclic AMP (cAMP) and prostaglandin E₂ (PGE₂) in macrophages. In vitro studies using mouse peritoneal macrophages demonstrated that GL at 50–100 µg/mL could significantly alter intracellular cAMP and PGE₂ levels, contributing to its broad innate immunoregulatory effects [362].

2.13.3. Effect on Immunocompromised Models

In MAIDS mice, which are susceptible to Candida albicans, GL administered intraperitoneally at a dose of 10 mg/kg/day for 7 consecutive days restored antifungal resistance by suppressing Th2 cytokines (IL-4, IL-10) and promoting Th1-polarized CD4⁺ T cells [363]. Similarly, in burn-injured mice, daily intraperitoneal administration of GL at 10 mg/kg for up to 5 days inhibited the development of suppressor T cells and induced contrasuppressor T cells, thereby improving immune responsiveness [364].

2.13.4. Effect on Dendritic Cell Maturation and Adaptive Immune Biasing

GL enhances dendritic cell (DC) maturation by upregulating surface markers such as CD40, CD86, and MHC-II, and by increasing IL-12 production. In the study, mouse splenic DCs were treated with GL at concentrations ranging from 50 to 100 μg/mL. These GL-treated DCs stimulated significantly stronger allogenic T cell proliferation in mixed lymphocyte reactions. The cytokine profile was skewed toward a Th1-type response, characterized by increased IFN-γ and IL-10 production and reduced IL-4 levels. These immunomodulatory effects indicate that GL promotes a Th1-biased adaptive immune response, which is critical for effective antiviral and antitumor immunity [365].

2.13.5. Effect on Antigen-Induced Histamine Release

Glycyrrhetinic acid (GA), the active metabolite of GL, inhibits histamine synthesis and release in dexamethasone-activated mast cells and P-815 mastocytoma cells at concentrations of 10⁻⁶ to 10⁻⁴ M. In vivo studies using guinea pigs demonstrated that oral administration of GA at 50 mg/kg significantly reduced antigen-induced histamine release. These findings suggest GA’s role as a mast cell stabilizer with potential anti-allergic effects [366].

2.13.6. Effect on Unique Regulatory T Cell Subtypes

GL can induce a distinct subset of CD4⁺ T cells known as anti-BI2T cells, characterized by their selective secretion of IFN-γ without concurrent production of IL-2, IL-4, or IL-10. The induction of these cells in vitro required the presence of macrophages and exposure to 100 µg/mL of GL, indicating a macrophage-dependent mechanism and a novel immune regulatory axis distinct from the classical Th1/Th2 paradigm [367].

2.13.7. Effect on Humoral Immune Response

GL has been reported to enhance antibody production in vitro when administered alongside polyclonal B cell activators such as LPS or pokeweed mitogen, which suggested its capacity to support humoral immunity in addition to its well-documented effects on cellular immune responses [368].

Schematic representation of the molecular mechanisms underlying GL’s immunomodulatory effects, illustrating its impact on antibody production, immune cells, and other molecular targets, is shown in Figure 4.

2.14. Effect of Glycyrrhizin on Arthritis

GL exhibits significant anti-arthritic activity across different types of arthritis, including osteoarthritis, intervertebral disc degeneration, rheumatoid arthritis, and collagen-induced arthritis, through its anti-inflammatory, antioxidant, and immunomodulatory properties. The key mechanisms involved include the inhibition of pro-inflammatory cytokines, suppression of major signaling pathways (PI3K/AKT, NF-κB, HMGB1–p38/JNK, and COX-2/TxA2), and the modulation of oxidative stress and autophagy.

2.14.1. Effect on Osteoarthritis

In an in vitro and in vivo study, GL was found to significantly reduce osteoarthritis (OA) progression by modulating inflammatory and catabolic factors in human chondrocytes. GL inhibited IL-1β-induced production of pro-inflammatory mediators such as NO, PGE2, TNF-α, and IL-6. It also downregulated the expression of COX-2, iNOS, MMP3, MMP13, and ADAMTS5, while preserving the cartilage matrix components aggrecan and collagen II. Mechanistically, GL suppressed PI3K/AKT phosphorylation and NF-κB activation. In a mouse OA model, GL administration protected against cartilage degradation [369]. Moreover, in a rat model of osteoarthritis, GL administered intra-articularly at doses of 2, 4, and 10 mg/kg for 12 weeks effectively ameliorated joint pain, cartilage degeneration, and inflammatory responses. At the 10 mg/kg dose, GL significantly reduced catabolic enzymes (MMP-1, MMP-3), inflammatory cytokines (IL-1β, IL-6, TNF-α), and cartilage degradation markers (PGE2, CTX-II), primarily by inhibiting HMGB1 and the TLR4/NF-κB signaling pathway [370].

In ex vivo human osteoarthritis chondrocytes, GL suppressed IL-1β-induced inflammation and cytotoxicity by downregulating HMGB1. It effectively reduced levels of PGE2, NO, pro-inflammatory cytokines, and MMPs, thereby improving cell viability and inflammatory balance in osteoarthritis chondrocytes [371]. In a cellular model of intervertebral disc degeneration, sometimes referred to as osteoarthritis of the spine, GL was shown to suppress IL-1β-induced apoptosis and inflammation in nucleus pulposus (NP) cells by targeting HMGB1, a pro-inflammatory mediator elevated in degenerated disc tissues. GL treatment (at a concentration of 100 μM) effectively downregulated HMGB1 expression and significantly inhibited activation of the p38 and p-JNK signaling pathways, thereby protecting NP cells from degeneration. These findings suggest the therapeutic potential of GL in managing spinal osteoarthritis by modulating HMGB1 activity and downstream inflammatory signaling [372]. It also ameliorated temporomandibular joint osteoarthritis in rats by suppressing the HMGB1-RAGE/TLR4-NF-κB/AKT signaling cascade. Both low (20 mg/kg) and high (50 mg/kg) doses attenuated cartilage degeneration and inflammation, indicating its disease-modifying potential in osteoarthritic conditions [373].

2.14.2. Effect on Rheumatoid Arthritis

A preclinical study using a collagen-induced arthritis model in Wistar rats assessed GL alone and in combination with platelet-rich plasma (PRP). GL treatment for 8 weeks significantly reduced levels of HMGB1, beclin-1, and LC3 mRNA, indicating reduced inflammation and autophagy. Antioxidant defense was enhanced via suppression of MPO activity and upregulation of catalase and Nrf2 DNA-binding activity. The combined GL + PRP group exhibited the most pronounced therapeutic effect [374]. Additionally, GL may target the COX-2/thromboxane A2 (TxA2) pathway, a crucial mechanism in rheumatoid arthritis (RA) pathogenesis. Since NSAIDs and DMARDs do not typically act via this route, GL presents a novel adjunct strategy to reduce inflammation and mitigate side effects of conventional RA therapies [375].

2.14.3. Effect on Collagen-Induced Arthritis

In a CIA rat model, GL (26.78 mg) administered in combination with triptolide (TP) (13.40 µg) significantly reduced the arthritic index, serum TNF-α, and anti-CII IgG levels. This combination therapy was as effective as a higher dose of TP alone (17.86 µg). Furthermore, IL-10 levels were notably elevated in the combination groups, indicating an anti-inflammatory immunoregulatory effect [376].

2.14.4. Effect on Osteoclastogenesis and Osteoporosis

GL was found to suppress osteoclastogenesis, a critical factor in bone loss. Li et al. [377] demonstrated that GL inhibited RANKL-induced osteoclast formation in vitro, reduced inflammatory cytokines (TNF-α, IL-1β, IL-6), and suppressed ROS production. Mechanistically, it activated the AMPK/Nrf2 pathway while inhibiting NF-κB and MAPK signaling, and upregulated antioxidant enzymes such as HO-1, NQO-1, and GCLC. These findings highlight GL’s therapeutic potential in managing osteoporosis and related bone disorders. Additionally, in a mouse model of periprosthetic osteolysis induced by PMMA particles, local treatment with GL significantly attenuated inflammatory bone loss. It did so by reducing osteoclast activity (TRAP+ cells), suppressing senescence-associated secretory phenotype (SASP) markers such as MIF and cathepsins, and enhancing senescence-protective markers including SDF-1, SIRT1, and SIRT6 [378]. In osteoporotic conditions, GL improved bone mass and microarchitecture in ovariectomized mice by suppressing RANKL-induced osteoclastogenesis and inactivating NF-κB signaling, thereby reducing expression of c-FOS and NFATc-1 [379].

2.15. Effect of Glycyrrhizin on Various Microbial Pathogens

2.15.1. Effect on Bacterial Pathogens

GL has demonstrated significant antibacterial activity across various pathogens and experimental models. In the case of multidrug-resistant (MDR) Pseudomonas aeruginosa strains (MDR9 and B1045), GL showed minimum inhibitory concentrations (MICs) of 40 mg/mL and 15 mg/mL, respectively. When co-administered with ciprofloxacin, GL reduced the MIC of ciprofloxacin from 32 µg/mL to 16 µg/mL. In a C57BL/6 mouse model of keratitis, this combination therapy reduced bacterial burden, MPO activity, and clinical severity scores more effectively than ciprofloxacin alone. The mechanism involved increased membrane permeability of the bacteria and downregulation of efflux pump activity [380]. In another antibacterial study, GL improved resistance to P. aeruginosa burn wound infections in thermally injured mice. The mechanism involved restoring β-defensin production in epithelial tissues by suppressing Gr-1⁺CD11b⁺ suppressor cells, which otherwise inhibit antimicrobial peptide synthesis via cytokines such as CCL2 and IL-10. GL-treated mice showed resistance to sepsis, while untreated controls did not survive the infection [381]. In dental microbiology, GL inhibited the glucosyltransferase enzyme of Streptococcus mutans in vitro, reducing glucan formation from sucrose and preventing bacterial adherence, a key step in dental plaque formation [382].

Studies using AGS gastric cancer cells and a mouse infection model demonstrated that Helicobacter pylori impairs autophagy by increasing lysosomal membrane permeabilization. Treatment with GL effectively restored lysosomal membrane integrity, enhanced autolysosome formation, and reduced the intracellular H. pylori load. Additionally, it alleviated gastric inflammation and tissue damage in infected mice. These findings suggest that glycyrrhizin, by inhibiting HMGB1, strengthens host autophagic defenses and holds promise as a potential antibacterial agent against H. pylori [383]. Moreover, GL at 5–10 mg/mL reduced adherence and enhanced early biofilm dispersal of MDR strains MDR9 and B1045 of Pseudomonas aeruginosa clinical isolates on corneal epithelial cells. Proteomic analysis revealed significant downregulation of antibiotic resistance, biofilm, and type III secretion proteins in MDR9. It also disrupts bacterial virulence and early biofilm formation, with strain-specific proteomic effects [384].

2.15.2. Effect on Fungal Pathogens

GL also exhibits significant antifungal properties. In a burn-induced immunosuppression model, thermally injured mice showed increased susceptibility to Candida albicans. Administration of GL (intraperitoneally) reversed this susceptibility by suppressing CD8⁺CD11b⁺TCRγδ⁺ type 2 T cells, which are responsible for immune suppression. GL also promoted CD4⁺ T cell activity, restoring antifungal immunity. Importantly, the adoptive transfer of CD4⁺ T cells from GL-treated donors conferred resistance to C. albicans in naïve mice. Moreover, blocking IL-4 and IL-10 cytokines using monoclonal antibodies produced similar results, supporting the mechanism of type 2 cytokine suppression [385].

2.15.3. Effect on Different Viruses

GL demonstrates potent, broad-spectrum antiviral activity by inhibiting viral replication, entry, and modulating host immune responses, with proven efficacy against viruses like HIV-1, influenza, and SARS-CoV/CoV2. Its low toxicity and multi-targeted mechanisms make it a promising adjunct or alternative antiviral agent [386,387,388,389]. The key findings from various antiviral studies on GL are summarized in

Table 2. Antiviral activity of glycyrrhizin across various preclinical and clinical models.

Virus	Model	Dose/Concentration	Key Findings	Mechanism of Action	References
HIV-1, R5 HIV	In vitro (MT-4, MOLT-4, PBM/MA); clinical (HIV+ patients)	0.075–0.6 mM (in vitro); 400–1600 mg/day IV (clinical)	Inhibits HIV replication; reduces p24 antigen; improves CD4+ counts and liver function; suppresses PKC and CCR5	Inhibition of protein kinase C; CCR5 suppression; β-chemokine induction; membrane stabilization	[390,391,392,393,394,395,396,397]
HSV-1, HSV-2, HHV-6/7, VZV	In vitro (Vero, HEp-2, rat CCEC, HEF cells); In vivo (burned/thermal injury mice)	0.5–3.6 mM (in vitro); 10 mg/kg (mice)	Inhibits replication, cytopathic effects, and pain; enhances resistance in burns; synergizes with acyclovir	Inhibits viral gene expression and adhesion; immunomodulation; contrasuppressor T cell induction	[364,394,398,399,400,401,402,403]
CMV	In vitro (U-937, MRC-5 cells); clinical (HIV+ child)	100 μg/mL (in vitro); 400 mg/day IV (clinical)	Suppresses CMV antigen expression; improves vision and reduced CMV recurrence in treated patient	Inhibits early viral gene expression; enhances host immunity	[404,405]
IFV (A, H1N1, H2N2, H5N1)	In vitro (A549, human lung cells); in vivo (BALB/c mice)	25–100 μg/mL; 10–50 mg/kg (mice)	Inhibits viral entry and replication; 100% survival with ribavirin; reduces cytokine storm	Inhibits endocytosis; suppresses ROS-NFκB/JNK/p38; induces IFN-γ	[406,407,408,409,410]
SARS-CoV	In vitro (clinical isolates of CoV (FFM-1 and FFM-2) from patients)	1000–4000 mg/L	Effective against SARS-CoV; blocked replication of the virus	Inhibits replication; ACE2 receptor interaction; cytokine modulation	[388]
SARS-CoV2 (S-RBD and Orf3a)	In vitro (Vero E6 cells; A549; NCI-H1299; BEAS-2B; AGS; A498); clinical (combination with boswellic acids 200 mg)	0.004–4 mg/mL; 1 mM; 60 mg (clinical)	Effectively inhibits virus replication; mitigates viral proteins-induced lung cell pyroptosis and activation of macrophages; inhibits binding of spike protein to ACE2; reduces mortality and recovery time of COVID-19 patients	Blocks viral replication via inhibition of viral main protease Mpro; down-regulates ACE2 expression; reduces excessive release of pro-inflammatory cytokines (IL-1β, IL-6, IL-8) and ferritin; modulates cytokine responses and reduces systemic inflammation.	[411,412,413,414,415]
PV, MeV, CHPV	In vitro (Vero and HeLa cells)	1.216 mM	Significant plaque reduction; broad antiviral activity	Inhibits viral adsorption and early replication	[398]
HTLV-I	In vitro (MT-2 cells)	~0.6 mM	Inhibits viral production and syncytia formation	Suppression of viral fusion and protein synthesis	[416]
FIV	In vitro (FL-4 cells)	0.15–0.6 mM	Reduces apoptosis in infected cells	Antioxidant and anti-apoptotic properties	[417]
Flaviviruses	In vitro (Vero cells, PS and HeLa)	100–1000 μg/mL	Inhibits replication across various flaviviruses (DENV-1–4, JEV, WNV, USUV, LGTV, YFV and WESSV)	Broad-spectrum antiviral; likely via membrane fluidity alteration	[418,419]
PEDV	In vitro (Vero cells)	0.1–0.8 mM	Inhibits viral entry and replication; reduces inflammation	Blocks HMGB1/TLR4-MAPK pathway	[420,421]
PRRSV	In vitro	200–800 μM	Inhibits viral penetration phase	Prevents virus entry post-adsorption	[422]
VSV	In vitro (kinase assays)	20 μM	Inhibits viral kinase-mediated phosphorylation	Inactivates viral kinase P	[423]
HBV	In vitro (HepG2 cells)	<400 µM	Inhibits secreted HBsAg and HBeAg; reduces replicative intermediates of HBV DNA and cccDNA	Acts as an HMGB1 inhibitor	[424]

Abbreviations: HIV—Human Immunodeficiency Virus; HSV—Herpes Simplex Virus; HHV—Human Herpesvirus; VZV—Varicella Zoster Virus; CMV—Cytomegalovirus; HTLV—Human T-cell Leukemia Virus; FIV—Feline Immunodeficiency Virus; JEV—Japanese Encephalitis Virus; SARS-CoV—Severe Acute Respiratory Syndrome Coronavirus; PEDV—Porcine Epidemic Diarrhea Virus; PRRSV—Porcine Reproductive and Respiratory Syndrome Virus; DHV—Duck Hepatitis Virus; VSV—Vesicular Stomatitis Virus; MeV—Measles Virus; PV—Poliovirus; CHPV—Chandipura Virus; IFV—Influenza Virus; DENV—Dengue Virus; WNV—West Nile Virus; USUV—Usutu Virus; LGTV—Langat Virus; YFV—Yellow Fever Virus; WESSV—Wesselsbron Virus, ZIKV—Zika Virus; HBV—Hepatitis B Virus; ID₅₀—50% Inhibitory Dose; IV—Intravenous; ROS—Reactive Oxygen Species; NF-κB—Nuclear Factor Kappa-light-chain-enhancer of activated B cells; MAPK—Mitogen-Activated Protein Kinase.

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2.16. Other Pharmacological Activities of Glycyrrhizin

2.16.1. Effects on Reproductive Health

In addition to its well-established pharmacological effects, GL has also exhibited a range of other therapeutic activities in various experimental models. Kumagai et al. [425] showed that GL inhibited estradiol-17β-induced uterine weight gain and β-glucuronidase activity in ovariectomized rodents. This effect was most pronounced at a glycyrrhizin–estradiol ratio of 1000:1, resulting in over 50% inhibition, while higher ratios reversed the effect. Importantly, GL alone had no estrogenic activity.

2.16.2. Effects in Aplastic Anemia

GL has also been explored for a variety of adjunctive therapeutic roles across different clinical settings. Notably, its immunomodulatory and hepatoprotective effects were demonstrated in a clinical study on non-severe aplastic anemia (NSAA), where Ren et al. [426] reported that co-administration of GL with cyclosporine A (CsA) significantly improved treatment response rates (82.86%) compared to CsA alone (60.61%), while also reducing liver toxicity, a common side effect of CsA. These findings underscore GL’s dual role in enhancing immunosuppressive efficacy and protecting against drug-induced hepatic injury.

2.16.3. Effect Against Chemotherapy-Induced Toxicity

Its cytoprotective potential is further supported by clinical studies showing GL’s ability to mitigate chemotherapy-related adverse effects. Akimoto et al. [427] demonstrated that a GL-based interferon-inducing formulation (SNMC) substantially reduced the toxicities associated with MMF (mitomycin, methotrexate, futraful) therapy in postoperative breast cancer patients, with a notably lower therapy discontinuation rate (1.7%) compared to controls (25%). Similarly, Kong et al. [428] observed that compound GL reduced the incidence of cytarabine-induced side effects, such as skin rash, fever, and cytarabine syndrome, without negatively impacting hematological parameters. These findings reinforce GL’s role as a supportive agent in reducing the toxicity burden of chemotherapeutic regimens.

2.16.4. Effects in Precocious Puberty

It demonstrated protective effects against precocious puberty (PP) in both human observational and rodent interventional models. In children, GL intake was associated with a significantly reduced risk of PP, especially in girls. Similarly, in danazol-induced PP rats, GL delayed puberty onset. Mechanistic studies revealed that GL modulated the gut microbiome, leading to suppression of the hypothalamic–pituitary–gonadal axis. Fecal microbiota transplantation further confirmed that gut microbiota alterations mediated GL’s protective effects against PP [429].

2.16.5. Effect on Oral and Periodontal Health

In another domain, GL has shown promise in oral health. A controlled clinical trial by Steinberg et al. [430], employing a split-mouth design in 21 dental students, found that topical GL application significantly reduced dental plaque formation within just 3 days, with more pronounced effects by day 4. Notably, these improvements occurred independently of dietary changes or oral hygiene routines. GL, when evaluated for its therapeutic effect against chronic periodontitis (CP) using TNF-α-treated human periodontal ligament stem cells (hPDLSCs) and a rat model of CP, attenuated periodontal inflammation by downregulating HMGB1 and associated pro-inflammatory cytokines. In the in vitro model, GL at concentrations of 1, 2, and 5 mM significantly inhibited TNFα-induced overexpression of HMGB1, IL-6, and IL-1β in hPDLSCs. In vivo, GL administration in CP rats markedly reduced the elevated levels of HMGB1, TNF-α, IL-6, and IL-1β in gingival crevicular fluid [431].

2.16.6. Antivenom Activity

Additionally, GL exhibits antivenom properties. Assafim et al. [432] demonstrated that GL, administered intraperitoneally at 180 mg/kg, effectively countered coagulopathy induced by Bothrops jararaca venom. It inhibited venom-mediated clotting, enzymatic hydrolysis, and platelet aggregation in vitro, and reduced thrombus formation by 86% in vivo. When combined with traditional antibothropic serum, a synergistic effect was observed.

3. Toxicity Studies

While GL possesses significant therapeutic value, it also exhibits notable toxicological potential, especially when used at high doses or over extended periods. One of the most clinically significant adverse effects is pseudoaldosteronism, a condition that mimics hyperaldosteronism. This arises from the inhibition of 11β-hydroxysteroid dehydrogenase type 2 by glycyrrhetinic acid, a metabolite of GL. The resulting excess cortisol activates mineralocorticoid receptors, leading to sodium retention, potassium loss (hypokalemia), and hypertension [433].

Numerous clinical studies have documented pseudoaldosteronism resulting from GL intake. Takegoshi et al. [267] reported a case of fluid retention and electrolyte imbalance following high-dose GL therapy. Similarly, Nishiyama et al. [434] described muscle weakness in a patient receiving oral GL, consistent with mineralocorticoid excess. These findings are supported by Fujiwara et al. [435], who observed hypokalemia and sodium retention in three diabetic patients treated with 40–200 mg/day of intravenous GL for chronic hepatitis. Their symptoms were more pronounced due to concomitant insulin use. Hoshiai et al. [436] reported life-threatening ventricular arrhythmias in a hypertensive woman treated with GL, with laboratory findings confirming suppressed renin and aldosterone levels and marked hypokalemia. Takabatake et al. [437] described congestive heart failure in a 66-year-old woman with hypertrophic cardiomyopathy after two weeks of 225 mg/day GL. Clinical signs included pleural effusion, pulmonary congestion, hypokalemia, and low renin-aldosterone levels. Notably, discontinuation of GL and bed rest alone resulted in full recovery.

Muscular and neuromuscular complications also emerge due to prolonged GL exposure. Shintani et al. [438] reviewed over 59 cases of hypokalemic myopathy resulting from chronic GL use, with symptoms including flaccid paralysis and severe muscle pain. Hayashi et al. [439] further investigated neuromuscular excitability in two patients receiving 270–273 mg/day GL. They developed hypokalemic myopathy with myotonic and repetitive discharges on electromyography, which resolved upon correction of serum chloride levels above 90 mEq/L. Some reports highlight even more severe neuromuscular outcomes. Iwasaki et al. [440] documented a case of GL-induced hypokalemic myopathy that progressed to respiratory arrest and laryngeal edema. Similarly, Kurisu et al. [441] analyzed multiple cases of symptomatic hypokalemia in elderly patients, reporting symptoms like fatigue, muscle weakness, and arrhythmias.

Toxicological effects are not limited to the endocrine and muscular systems. Rossi et al. [442] demonstrated cardiotoxic effects of 18α-glycyrrhetinic acid in animal models, including myocardial edema, myolysis, and apoptosis, alongside significant ECG changes. Experimental research by Sobotka et al. [443] further showed that ammoniated GL administered to rats for 4–6 months induced hypertension, bradycardia, polydipsia, and behavioral changes, particularly affecting conditioned avoidance responses. Its impact extends to hepatic drug metabolism as well. Paolini et al. [444] demonstrated that repeated administration of licorice extract or GL in mice significantly induced liver cytochrome P450 enzymes, particularly CYP3A, which altered testosterone metabolism and increased the risk of drug interactions. This enzyme induction could affect the pharmacokinetics and toxicity profiles of concurrently administered medications. Recent studies have identified biomarkers associated with GL toxicity. Takahashi et al. [445] found that elevated serum levels of 18β-glycyrrhetyl-3-O-sulfate were correlated with decreased potassium, renin, and aldosterone levels, confirming its role in the development of pseudoaldosteronism.

Due to these wide-ranging toxicological effects, there is growing concern about high-dose GL consumption. The German Research Foundation’s Senate Commission on Food Safety has emphasized the need for safe intake limits and regulatory oversight. Eisenbrand [446] reported the Commission’s position that GL’s potential to disrupt electrolyte balance warrants caution in its use in foods, supplements, and herbal medications.

These studies show that GL can affect several body systems, such as the hormonal, heart, nerve, liver, and possibly brain systems, making it important to monitor its dose, especially in older adults and people with existing health problems.

4. Pharmacokinetic Studies

Several studies have investigated the pharmacokinetics of GL, revealing a dose-dependent and nonlinear disposition profile. In rats, intravenous administration at doses of 5, 10, 20, and 50 mg/kg showed a biexponential decline in plasma concentrations, with the higher doses (20 and 50 mg/kg) resulting in a more than proportional increase in the area under the curve (AUC). This suggests saturation of metabolic or excretory pathways at elevated doses. Additionally, higher doses led to a significant increase in the steady-state distribution volume and a reduction in both total body clearance and biliary clearance, demonstrating altered drug distribution and elimination dynamics [447,448]. In a mouse model, Shibata et al. [449] examined the absorption and disposition of GL delivered via an intestinal pressure-controlled colon delivery capsule. Following intravenous administration of 10 mg/kg, plasma GL levels declined rapidly at first and then more gradually. Interestingly, intracolonic administration (50 mg/kg) resulted in significantly higher systemic absorption compared to intraduodenal or intravenous administration, highlighting the potential of colon-targeted delivery systems to enhance oral bioavailability.

Further pharmacokinetic analysis by Koga et al. [450] demonstrated the impact of serum albumin on GL disposition. In rats with albumin deficiency, GL showed reduced serum protein binding at 2.5 μg/mL concentration. As biliary excretion is the rate-limiting step in GL elimination, these findings suggest that therapeutic efficacy may be preserved even in chronic hepatitis patients with hypoalbuminemia, as the lower binding does not compromise drug elimination. Zhong et al. [451] investigated GL’s biotransformation using an in situ vascularly perfused rat intestine-liver model. The study confirmed that GL is extensively metabolized by gut microbiota and liver cells, while minimal metabolism occurs in intestinal mucosa cells. Extraction ratios were reported as 4.2% in the intestine and 28.0% in the liver. The absorption rate constant was calculated at 0.33/min, and glycyrrhetinic acid, the active metabolite, was detected in both perfusate and intestinal luminal fluid following intraduodenal administration.

Human pharmacokinetic data are also available. Kočevar Glavač and Kreft [452] administered 600 mg of GL to six healthy volunteers and reported that repeated ingestion of such high doses could lead to adverse effects such as pseudoaldosteronism. This toxicity is attributed to the GL metabolite, 3β-monoglucuronyl-18β-glycyrrhetinic acid (3-MGA). The urinary excretion of 3-MGA ranged from 1425.9 to 3147.8 μg, representing 0.31–0.67% of the ingested dose, with elimination rates of 31.52–209.47 μg/h. Notably, the time of ingestion (morning vs. evening) did not significantly affect metabolite elimination. Moreover, it significantly influences drug pharmacokinetics and is associated with a range of clinically relevant drug–drug interactions due to its modulation of metabolic, absorptive, and eliminative pathways. One of the key pharmacokinetic effects of GL is its ability to inhibit the metabolic clearance of drugs. In a clinical study, co-administration of GL with prednisolone (PSL) led to elevated plasma levels of both total and free PSL, along with increased AUC and mean residence time (MRT), while clearance was reduced, though volume of distribution remained unchanged. This indicates that GL can slow the metabolic degradation of PSL, potentially amplifying both its therapeutic and adverse effects [453].

In addition to inhibitory effects, GL has also been shown to induce cytochrome P450 enzymes, particularly CYP3A. Chronic ingestion of GL in humans resulted in decreased plasma concentrations of midazolam, a well-known CYP3A substrate [454]. Similarly, in rats, GL pre-treatment significantly enhanced the metabolism of triptolide (TP), lowering its AUC, half-life, and MRT. These changes were reversed upon co-administration of ketoconazole, a CYP3A inhibitor, confirming the role of GL in inducing CYP3A activity [455].

GL and its active metabolite, 18β-GA, also influence intestinal drug absorption by interacting with P-glycoprotein (P-gp), a key efflux transporter. Studies using Caco-2 cell monolayers and in vivo rat models demonstrated that 18β-GA enhances the bioavailability of low-absorption drugs like paeoniflorin and calcitonin by reducing the intestinal barrier resistance and increasing membrane permeability [456,457,458]. However, in contrast, some studies have reported that GL increases the efflux ratio of drugs such as puerarin and nobiletin, leading to a reduction in their systemic exposure. This is likely due to P-gp induction or enhanced hepatic metabolism, and was associated with decreased AUC, Cmax, and half-life, emphasizing the need for dose adjustment when such compounds are co-administered with GL [459,460].

Additionally, 18α-glycyrrhizin has been shown to potentiate the effect of glibenclamide in diabetic rats by decreasing its elimination rate and increasing its pharmacokinetic parameters such as Cmax, AUC, and half-life. This effect appears to be largely due to inhibition of CYP3A [461]. Collectively, these findings highlight the complex and bidirectional pharmacokinetic behavior of GL: it can both inhibit and induce drug-metabolizing enzymes like CYP3A and affect drug absorption through modulation of intestinal P-gp activity. These interactions have significant implications for drug efficacy and safety, underscoring the importance of exercising caution when GL is co-administered with drugs that have narrow therapeutic windows or are extensively metabolized by the liver.

Hence, pharmacokinetic evidence from previous studies demonstrates the clinical importance of GL in drug therapy. Its nonlinear disposition, extensive gut–liver metabolism, and ability to modulate CYP3A enzymes and P-glycoprotein can either enhance or reduce the exposure of co-administered drugs, resulting in clinically significant drug–drug interactions. These findings underscore the need for cautious dose optimization and close monitoring, particularly when GL is combined with drugs that have narrow therapeutic windows or undergo extensive hepatic metabolism.

5. Conclusions and Future Perspectives

GL demonstrates a wide array of pharmacological activities, including anti-inflammatory, antiviral, hypertensive, hepatoprotective, antidiabetic, immunomodulatory, and antitumor effects, all supported by substantial experimental and clinical evidence [462,463,464,465,466,467,468]. These therapeutic benefits are largely mediated through the modulation of key molecular pathways such as NF-κB, MAPK, PI3K/Akt, and cytokine signaling [469,470,471]. Clinically, GL has shown notable efficacy in liver diseases and viral infections by suppressing immune-mediated tissue damage and inhibiting viral replication [472,473,474,475]. Its therapeutic scope also extends to cancer, neurodegenerative diseases, cardiometabolic disorders, and gastrointestinal conditions, making it a promising candidate for the management of multifactorial diseases.

Despite its promising pharmacological profile, GL’s clinical utility is limited by poor oral bioavailability, significant interindividual variability in metabolism, largely due to gut microbial processing, and potential side effects such as pseudoaldosteronism. In this condition, GL metabolites inhibit 11β-HSD2, blocking cortisol inactivation. The resulting cortisol excess activates mineralocorticoid receptors, causing sodium and water retention, potassium loss, hypertension, edema, and hypokalemia [476]. Additionally, the two epimeric forms (18β and 18α) differ in stability, biological activity, and pharmacokinetics, making epimerization an important consideration in drug development. The conversion of 18β-GL to the more thermodynamically stable 18α-GL can be deliberately induced during purification or processing, but may also occur as an unintended side reaction [477,478]. These limitations highlight the need for strategic innovations to improve its therapeutic performance and safety. Hence, future research should prioritize the design of GL analogs or derivatives with improved bioavailability and reduced toxicity to overcome these challenges.

Structural modifications, particularly to the sugar moieties or the glycyrrhetinic acid backbone, hold potential to enhance GL’s pharmacodynamic specificity and therapeutic potency. Previous studies clearly indicate that the CNS effects of orally administered GL are largely mediated by its metabolite, 18β-glycyrrhetinic acid (18β-GA), formed through gut microbial metabolism due to GL’s poor oral bioavailability. Unlike GL, 18β-GA readily crosses the blood–brain barrier and has been detected in both brain tissue and cerebrospinal fluid, making it the primary bioactive form responsible for GL’s neuroprotective and anti-neuroinflammatory effects [479]. Furthermore, advances in nanotechnology provide additional opportunities to overcome GL’s pharmacokinetic limitations. Nanoformulations such as GL-loaded liposomes, micelles, and nanoparticles have demonstrated improved solubility, stability, and targeted tissue delivery, particularly in liver and lung diseases. Continued refinement of these delivery systems may significantly broaden GL’s therapeutic potential, including its use in CNS disorders.

Despite encouraging preclinical and clinical findings of GL, comprehensive clinical validation remains a priority. Large-scale, randomized controlled trials across diverse populations are essential to confirm efficacy, determine optimal dosing strategies, and assess long-term safety. Equally important is the standardization of extract compositions and formulation protocols to ensure reproducibility and regulatory compliance. Moreover, its well-documented immunomodulatory activity also makes it a strong candidate for combination therapies, potentially enhancing outcomes in chronic inflammatory diseases, cancers, and viral infections through synergistic mechanisms. Importantly, its neuroprotective effects are observed at pharmacological doses, whereas mineralocorticoid-related adverse effects are typically linked to chronic, high-dose intake. Strategies such as controlled dosing, colon-targeted delivery, and careful monitoring of serum potassium and blood pressure can help maximize therapeutic benefits while minimizing risks.

In addition to the aforementioned areas, integrating personalized medicine approaches, such as pharmacogenomic profiling and microbiome analysis, into future GL research could prove highly beneficial. These tools enable the customization of treatments based on individual metabolic variations, thereby enhancing both safety and therapeutic efficacy.