Rheum tanguticum Maxim. ex Balf. is a perennial plant belonging to the genus Rheum L. of the Polygonaceae family [1]. Its roots, known as Daehwang (or Da Huang in Chinese), have a long history of medicinal use in China, Korea, and Japan [2]. In Korea, Daehwang is clinically administered for a range of conditions, including constipation, abdominal pain, diarrhea, jaundice, nosebleeds, conjunctivitis, sore throat, appendicitis, bruises, amenorrhea, and burns [3]. Formulations such as Daeseunggitang (Da Cheng Qi Tang), Dohaekseunggitang (Taohe Chengqi Tang), Jeodangtang (Di Dang Tang), and Sasimtang (San Huang Xie Xin Tang) all incorporate Daehwang. Various neurological symptoms, including perceptions of seeing a ghost, experiences related to mental illness, and chest tightness, are included in the Chinese clinical guidelines, Shanghanlun [4]. Existing literature consists of retrospective case reports focusing on the aftermath of stroke, depression [5, 6], and schizophrenia [7]. Recently, preclinical research regarding the efficacy of R. tanguticum in addressing various neurological disorders has been conducted. Rhubarb anthraquinones, such as emodin, aloe-emodin, chrysophanol, and physcion, are known to protect the central nervous system [8]. Daehwang has also been found to exert anti-neuroinflammatory and neuroprotective effects by inhibiting microglial activation [9]. A study employing the Morris water maze indicated that treatments using R. tanguticum could attenuate cognitive dysfunction in a rat model of Alzheimer’s disease [10]. Emodin, one of the anthraquinones, improved kainic acid-induced epileptic rats through regulation of multidrug resistance gene 1 [11]. Therefore, R. tanguticum shows promise both as an antiepileptic and a neuroprotective agent in the treatment of epilepsy and neurodegenerative conditions.

Epilepsy is a neurological disorder characterized by recurrent seizures arising from various etiological factors and complex initiation mechanisms [12]. Factors such as congenital brain anomalies, abnormal neuronal migration, birth trauma, intracerebral hemorrhage, intracranial inflammation, febrile seizures, hypoxia, hypoglycemia, hypocalcemia, brain injury, intracranial tumors, and cerebrovascular diseases are attributed to the onset of epilepsy [13]. In Korea, the incidence of epilepsy ranges from 50 to 70 cases per 100,000 individuals annually, ranking it as the third most prevalent neurological condition after dementia and stroke. Within 5 years after an initial seizure, approximately 80% patients are likely to experience a subsequent seizure, evolving into chronic active epilepsy. Roughly 30% of patients with epilepsy exhibit drug resistance [14, 15]. Consequently, the mean disease duration spans 10 years, with 20–30% of cases manifesting as lifelong conditions. Considering these circumstances, there is a pressing need for innovative therapeutic approaches for seizure management. Following the emergence of the epilepsy-epileptogenesis paradigm, the focus has shifted toward understanding the mechanistic links between inflammation and epilepsy [16, 17, 18]. In this context, our research aims to investigate the efficacy of natural products in trimethyltin (TMT)-induced epilepsy models.

The organotin compound, TMT exhibits specific toxicity in the hippocampus, an essential part of the brain’s limbic system [19]. TMT exposure is associated with a range of neurological symptoms, such as hypoactivity evolving into hyperactivity, hyperexcitability, ataxia, tremors, seizures, convulsions, memory deficits, and learning impairments, collectively termed TMT syndrome [20, 21]. Consequently, TMT is used to simulate neurodegenerative conditions, such as epilepsy and dementia in animal models [22, 23], and the hippocampal damage closely resembles that caused by convulsant agents or observed in certain human epilepsy cases [24]. The neurotoxic effects of TMT are attributed to multiple mechanisms, including apoptotic cell death, calcium dyshomeostasis, oxidative stress, and neuroinflammation [25]. Much like kainic acid, commonly used to study temporal lobe epilepsy and status epilepticus, TMT induces neuronal loss in the CA3 and CA1 regions of the hippocampus [26]. This loss is thought to result from either glutamate-dependent excitotoxicity or calcium overload [23]. When glutamate receptor antagonists are co-administered with TMT, excitotoxicity is reduced, suggesting potential neuroprotective benefits [27]. Therefore, TMT is a valuable tool for generating animal models of neurodegeneration associated with cognitive decline and temporal lobe epilepsy. This is because the neurodegenerative effects of TMT share key pathogenic features common to a broad array of neurodegenerative disorders, such as selective neuronal death and neuroinflammation [23, 28].

The onset of symptoms in neurodegenerative diseases is frequently linked to neuroinflammation. Proinflammatory elements, including activated glial cells and microglia, have garnered significant interest as potential therapeutic targets for individuals with epilepsy [29]. An analysis of the dementia drug development pipeline in 2023 delineated the roles of candidate therapies aimed at amyloid, epigenetics, inflammation/immunity, metabolism/bioenergetics, neurogenesis, neurotransmitter receptors, and oxidative stress [30]. Therefore, this study sought to assess the regulatory efficacy of R. tanguticum in animal models of seizures and hippocampal neurodegeneration induced by TMT.

Identification of complex molecular pathways remains a substantial challenge in the context of herbal medications. Unlike single-compound agents, herbal medications usually comprise multiple components, complicating the task of elucidating specific action mechanisms [31]. Network pharmacology has recently emerged as a new approach for identifying compound-target pathways related to particular diseases, offering a systematic and holistic perspective. Despite advances in clarifying the actions of various herbs and pharmacological agents through network pharmacology’s “multi-target–multi-pathway” frameworks, the molecular mechanisms accounting for the effectiveness of R. tanguticum in epilepsy treatment remain undefined.

In this study, the therapeutic potential of R. tanguticum for epilepsy was evaluated by integrating experimental research with network pharmacology. Initially, a mouse model was employed to investigate the protective effects against TMT-induced hippocampal degeneration. Subsequently, a network pharmacological analysis was conducted to comprehensively evaluate the regulatory mechanisms involved in epilepsy treatment, focusing on potential active compounds and target genes (Fig. 1).

Fig. 1.

Flowchart for investigating therapeutic potential of Rheum tanguticum in epilepsy treatment. TCMSP, traditional chinese medicine systems pharmacology database and analysis platform; HIT, herbal-ingredient-target platform; BATMAN-TCM, bioinformatics analysis tool for molecular mechanism of TCM; TTD, therapeutic target database; PPI, protein-protein interaction; KEGG, Kyoto encyclopedia of genes and genomes; GO, gene ontology; TMT, trimethyltin; RTE, Rheum tanguticum root extract; TNF-$\alpha$, tumor necrosis factor $\alpha$; GFAP, glial fibrillary acidic protein; CON, control; Iba-1, ionized calcium-binding adaptor molecule 1; SWISS, SwissTargetPrediction. Results are expressed as mean $\pm$ standard error. **p 0.01, ***p 0.001 versus the controls. ${}^{\mathrm{\#}}$p 0.05, ${}^{\mathrm{\#}\mathrm{\#}}$p 0.01, ${}^{\mathrm{\#}\mathrm{\#}\mathrm{\#}}$p 0.001 versus the TMT group.

The primary objective of pharmacotherapy in the management of epilepsy is to achieve complete remission of epileptic seizures. Medications targeting the reduction of neuronal excitation or the augmentation of inhibitory processes through various mechanisms, such as gamma-aminobutyric acid (GABA)-receptor enhancement, inhibition of glutamate excitation, or the modulation of voltage-gated sodium and calcium channels, are widely utilized [54]. Nevertheless, studies indicate that over 30% persons with epilepsy exhibit resistance to antiepileptic drugs [55]. Furthermore, the prolonged use of these medications is often associated with somatic, neurological, psychiatric, and neonatal adverse effects, posing significant challenges to epilepsy pharmacotherapy [56]. This situation underscores the urgent need for research regarding natural substances that possess antiepileptic properties through novel mechanisms.

Our study demonstrates that RTE mitigates the severity of TMT-induced epileptic seizures and associated weight loss in a dose-dependent manner (Fig. 3B,C). R. tanguticum, which is one of the source plants for Daehwang, a recognized medicinal in China, Korea, and Japan, was utilized in this investigation. Hepatotoxicity of Daehwang has been previously reported [57, 58], so clinical use requires toxicity control, such as compliance with appropriate dosage and use of hot water extraction method. The recommended dosages for Daehwang are documented in the pharmacopeias of these nations, and it is available commercially as a raw component for herbal preparations. The neuroprotective effects of R. tanguticum as a part of the formulation, Geijigadaehwang-tang has been studied previously [32], and our findings confirm its antiepileptic and neuroprotective potential. However, variations in the therapeutic effects may arise depending on the geographical location of the source and processing of the plants used as Daehwang, which warrants further research. Moreover, the chronic toxicity assessments of rhubarb extract have indicated a NOAEL of 94 mg/kg/day, reflecting a range of biological safety in several contexts [35]. Our findings showed no significant change in body weight for dosages from 10 to 100 mg/kg/day, with RTE notably inhibiting the weight loss associated with TMT-induced neurotoxicity (Fig. 3B).

Therefore, we conducted a component analysis to verify the quality standards and identify the constituents of R. tanguticum. Sennoside A, sennoside B, chrysophanol, emodin, physcion, (+)-catechin, and quercetin-3-O-glucuronide were identified as components in RTE by UPLC (Fig. 2). These findings conform to the results of the component analyses of rhubarb and Daehwang performed in previous studies [57, 58]. Various research efforts have underscored the neuroprotective properties of these constituents. For example, sennoside derivatives have shown potent antioxidative effects against oxidative stress induced by hydrogen peroxide, light, and radiation, mitigating the production of reactive oxygen species production and neutralizing free radicals [59, 60]. Chrysophanol has been noted for its efficacy in reducing inflammation in focal ischemic brain injuries and decreasing proinflammatory cytokines, including TNF-$\alpha$ and Interleukin-1 beta (IL-1$\beta$) [61]. Emodin has been recognized for its potential to reduce neurotoxicity in numerous neurological disorders, such as anxiety, depression, cerebral ischemia, Alzheimer’s disease, Parkinson’s disease, and schizophrenia [62]. Physcion has been validated to confer protection against neuronal damage from cerebral ischemia-reperfusion by inhibiting the Toll-like receptor 4/nuclear factor kappa-light-chain-enhancer of activated B cells (TLR4/NF-$\kappa$B) pathway [63]. Catechin is widely recognized for its neuroprotective effects and has been extensively investigated for its antioxidant and iron-chelating properties, which protect against neurodegenerative diseases like Parkinson’s disease and Alzheimer’s disease [64]. Finally, quercetin-3-O-glucuronide has been found to protect neurons from Parkinson’s disease in animal models, such as the mouse 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and rat 6-hydroxydopamine (6-OHDA) models, by suppressing reactive oxygen species and hindering apoptosis [65, 66].

Additional active compounds, including (-)-catechin, sennoside D, sennoside E, and aloe-emodin, which are isomers of those verified by the UPLC technique were identified by the in silico PPI analysis (Table 2). The compound, rhein is primarily known for its neuroprotective effects, which are attributed to its antioxidant and anti-inflammatory properties [67, 68]. Similarly, baicalein has been shown to be effective in a range of epilepsy models, including temporal lobe and posttraumatic epilepsy ones [69, 70]. Owing to the lack of available standards, only a subset of the RTE components were identified by UPLC. However, compounds similar to rhein and baicalein, though not directly identified, may be present in RTE. In silico methods help estimate the other RTE constituents that may contribute to seizure inhibition and neuroprotection. Therefore, despite discrepancies between computer simulations and component analysis, both strands of evidence corroborate the therapeutic potential of R. tanguticum root in treating epilepsy.

TMT is known to be associated with neurodegeneration in the central nervous system, especially in the hippocampus. The pathogenic mechanisms of TMT involve several processes, including oxidative stress, neuroinflammation, neuronal death, and regeneration [25]. While the precise mechanisms remain unclear, glutamate-mediated excitotoxicity and calcium dysregulation by TMT play important roles in the pathophysiology of epilepsy [71]. In our study, mice exposed to TMT exhibited aggressive behavior and seizures, which resemble symptoms of degenerative brain disorders [28]. When RTE was administered to these mice, an amelioration of these clinical symptoms was observed on the first and second days after treatment (Fig. 3B,C).

DCX is found in proliferating neural progenitors and neuronal precursors during adult neurogenesis and serves as a reliable marker for neurogenesis in adult brains [72]. Adult mouse hippocampi typically exhibit high DCX levels. However, a previous study showed that DCX expression markedly reduced after a 2–4-day exposure to TMT, with notable recovery observed on the 10th day after exposure [73]. We found that adult neurogenesis significantly reduced 2 days after TMT treatment, although RTE administration prevented this neuronal degeneration (Fig. 3D,E). Additionally, neuronal death has been identified as a critical factor in epileptogenesis [52]. Our study suggests that RTE treatment mitigates TMT-induced neuronal degeneration and epilepsy-like behavior.

C-fos is recognized as a marker of neural activity and is associated with cellular development, differentiation, transformation, and death [74, 75]. After a seizure, c-fos activation is noticeable in the brain [76]. Previous studies have shown increased c-fos expression following TMT administration, which is correlated with the intensity of seizure behaviors that was highest 2 days after administration [38]. In our study, RTE lowered the mRNA expression of c-fos, thus mitigating seizure behaviors (Fig. 4A). Additionally, within the PPI network, both TNF (degree = 15) and Protein c-fos (FOS, degree = 13) were identified as central proteins in the context of epilepsy treatment (Fig. 6).

TNF-$\alpha$ is a major inflammatory regulator capable of inducing further cytokine production and potential neuronal death [77]. It may precipitate neuroinflammation and promote DG cell death in the TMT model [78, 79]. TMT exposure causes the activation of astrocytes and significant release of inflammatory cytokines, such as TNF-$\alpha$, which are detrimental to the brain [80]. We found that RTE treatment reduced the production of both TNF-$\alpha$ and GFAP (Fig. 4B,C), thereby offering protection against TMT-induced neuronal inflammation in the hippocampus. Consequently, our in vivo and in silico results suggest that proteins such as FOS and TNF play significant roles in neuroprotective activity.

The morphological analysis of the PPI network extended beyond c-fos, TNF-$\alpha$, and GFAP, and critical RT proteins implicated in seizures, including Interleukin (IL)-6, RAC-alpha serine/threonine-protein kinase (AKT1), Mammalian target of rapamycin (mTOR), and IL-1$\beta$, were identified (Table 3). Both experimental and clinical research have underscored the pivotal role of cytokines, including IL-6 and IL-1$\beta$, in the underlying pathophysiological mechanisms of epilepsy [81, 82]. Moreover, mTOR is recognized as a fundamental component of nervous system functionality, and mammalian target of rapamycin complex 1 (mTORC1) and 2 (mTORC2) function together to regulate neural network activity, dendritic architecture, synaptic function, and generation of new neurons, all of which are essential for cortical brain development [83]. Collectively, our integrated approach, combining experimental study and network pharmacology, highlights the potential of R. tanguticum to confer neuroprotection in the management of epilepsy by modulating proteins and pathways associated with neurodegeneration.

The implications of this study for clinical practice are indirect and preliminary, offering foundational insights for translational research in treating neurodegenerative conditions. While we did not investigate the effect of varied concentrations of Daehwang extract for extended treatment durations, our findings lay the groundwork for subsequent experimental investigations involving these compounds.

In summary, this study identified the components of RTE and demonstrated its in vivo anti-toxic activity against TMT damage. Concurrently, RTE was observed to attenuate the clinical symptoms of seizures, reduce hippocampal neurogenesis, and increase glial activation. Complementary to these findings, network pharmacology approaches revealed that 19 active compounds in RTE, interacting with 35 distinct targets, are integral to its antiepileptic efficacy. These compounds appear to exert their beneficial effects by modulating pathways associated with neurodegeneration and TNF signaling. Collectively, our findings suggest that RTE may possess therapeutic promise for conditions such as epilepsy and neurodegenerative diseases by counteracting inflammatory and oxidative pathways.

Data generated or analyzed during this study are included in this published article.

JC, SL, JSK, and SIL conceived and designed the experiment. SK, MNT, SMR, DHK, YEL, SWC, and SJ performed the experiments. JC, SMR, CM, SL, and JSK analyzed and interpreted the data. JC, SMR, JSK, CM, and SIL contributed reagents, materials, analysis tools or data. JC, SK, JSK, MNT, and SIL wrote the paper. SWC, JSK, and SIL wrote the review and editing. SWC acquired grant funding. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

All experimental procedures were conducted in accordance with the protocols approved by the Institutional Animal Care and Use Committee at Dongshin University (Approval No.: DSU2023-04-03).

Not applicable.

This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. 2021R1A2C1009604).

The authors declare no conflict of interest. Changjong Moon is serving as one of the Editorial Board members/Guest editors of this journal. We declare that Changjong Moon had no involvement in the peer review of this article and has no access to information regarding its peer review. Full responsibility for the editorial process for this article was delegated to Jesús Pastor and Gernot Riedel.