Chemical composition and anticonvulsant activities of herb pair of Gastrodia elata Blume-Acorus tatarinowii Schott decoction on experimentally induced seizures in mice

Epilepsy is a serious public health problem in the world. At present, over 30% of affected patients remain refractory to currently available treatment. Medicinal plants as pharmaceuticals and healthcare treatments have been frequently used in the management of epilepsy in China for many centuries. Gastrodia elata-Acous tatarinowii (GEAT), as a classic and most commonly used herb pair in traditional Chinese medicine (TCM), has been employed to control seizures for thousands of years. However, the animal experiment data on its anticonvulsant effect is limited in the literature. Thus, this study aimed to reveal the therapeutic actions of GEAT decoction against seizures in mice. UHPLC-MS/MS was performed to analyze the chemical components of GEAT decoction. The mice were given GEAT decoction for 7 days, and MES, PTZ, and 3-MP injection was given 30 min after the last administration. Video monitoring was performed for comparisons. In addition, the PTZ-induced kindling models were conducted to investigate the seizure severity, anxiety and cognitive profile, inflammation, and oxidative stress parameters in mice. The results showed that GEAT decoction dose-dependently protected mice against MES, 3-MP, and PTZ-induced acute seizures. Furthermore, GEAT decoction significantly ameliorated seizure severity, decreased the accumulation of inflammatory mediators TNF-α, IL-1β, and IL-6, mitigated oxidative stress, as well as alleviated anxious-like behavior and cognitive deficits in PTZ-kindled mice. These results suggest that GEAT decoction possesses certain anticonvulsant properties, which might be clinically useful as phytotherapy alone or as an adjunct therapy for the prevention and treatment of seizures and epilepsy.


Introduction
Epilepsy is a common and complex neurological disease, affecting more than 70 million people around the world (Thijs et al. 2019). Epilepsy has become a major concern in developing countries which affects approximately 80% of people (Khattak et al. 2021). Statistics show that epilepsy affects an estimated 12 million people in India (Uthara et al. 2017). In China, it is estimated that there are more than 12 million patients with epilepsy, of which about 8 million patients with active epilepsy are getting the drug (China Association against Epilepsy 2015; Ding et al. 2021). The prevalence and incidence of epilepsy are slightly higher in males than that in females (Beghi 2020). In addition, its incidence has a bimodal distribution with the highest risk in children and the elderly (Thijs et al. 2019). With the aging of the population increasing dramatically, the prevalence of epilepsy diseases is increasing rapidly in many countries in the future. Focal seizures are more common than generalized seizures both in children and in the elderly (Beghi 2020). Compared with elderly patients with epilepsy, the pathogenesis of epilepsy in childhood is more complex and diverse. There is a considerable part of childhood with epilepsy progressing to intractable epilepsy. Extensive research has demonstrated that intractable epilepsy is caused by numerous precipitating factors and can produce a great impact on the cognitive mental, psychological, and social functions of childhood with epilepsy (Hwang et al. 2019). Seizures might be the prominent feature in inherited metabolic disease (Hundallah and Tabarki 2021). Due to the poor understanding of the pathogenesis and the lack of significant therapeutic regimens, a difficult problem exists, therefore, for more effective therapies or alternative approaches to effective treatment-intractable epilepsy management (Löscher and Klein 2020). Therefore, the research on epilepsy and its treatment have extremely important practical significance and urgency.
At present, the major choice for the treatment of epilepsy in the clinic still relies on drugs . Despite the existing clinical antiepileptic drugs (AEDs) producing satisfactory seizure control for about 2/3 of epileptic patients, these available AEDs fail to control epileptic activity in about 1/3 of epileptic patients Kondrat-Wróbel and Łuszczki 2018;Bai et al. 2019). Currently available antiepileptic drugs can also not prevent the development of epilepsy drug resistance, which is considered to be a challenge in epilepsy treatment. In addition, current available AEDs only target symptoms but cannot prevent the underlying natural epileptogenesis and prognosis of epilepsy. With the worldwide and long-term use of these AEDs, their adverse effects have gradually emerged (Golyala and Kwan 2017;Silva et al. 2019;Li et al. 2020). For example, some common side effects including dizziness, headache, drowsiness, and attention deficit disorder, as well as cardiovascular abnormalities, hematotoxicity and heart damage, endocrine disorders, and suicide risk, etc. have been emphasized . Besides, large doses of antiepileptic drugs using may have harmful effects on intellectual development or language function (Golyala and Kwan 2017;Silva et al. 2019). What might be a solution to the problems facing drug resistance and side effects, those traditional Chinese medicine or botanical drugs that have been used for a long time have gradually drawn the attention of drug developers and researchers in recent years (Lin and Hsieh 2021;Khattak et al. 2021). For example, the natural components cannabidiol extracted from Cannabis sativa L. has been approved by FDA for the treatment of Lennox Gastaut syndrome and Dravet syndrome in children with refractory epilepsy (Mitelpunkt et al. 2019).
TCM has a long history in the treatment of epilepsy, which was recorded in the classical masterpieces Inner Canon of Huangdi (黄帝内经, in Chinese) as early as 2200 years ago. In particular, these records revealed the national characteristics and unique advantages of traditional Chinese herbs in the treatment and control of seizures in children (Bai et al. 2019). From the classic and traditional medicine point of view, the representative herbal or ethnic medicine widely used for treating seizures and epilepsy in TCM mainly included G. elata, A. tatarinowii, Arisaema heterophyllum Blume and Polygala tenuifolia (Xiao et al. 2015;Zhao et al. 2018;Bai et al. 2019). It has been reported that G. elata and A. tatarinowii with the most prominent effect used most frequently in the treatment of intractable epilepsy in children (Bao et al. 2012). In addition, the Dingxian pill recorded in Yi Xue Xin Yu and Dianxian Kang capsule approved by CFDA as well as other commonly used drugs for the treatment of epilepsy mainly contain these two herbs. In these prescriptions, G. elata has the function of expelling wind and relieving convulsion, and A. tatarinowii makes expectoration easy and relieves mental stress. In our previous studies, the α-asaronol from A. tatarinowii decreased the severity of seizures in mice models of epilepsy, showing a broad spectrum of anticonvulsant activity Jin et al. 2020). Considering the compatibility mechanisms of formulas in TCM, the current study aimed to evaluate the anticonvulsant activities of GEAT decoction against seizures using electric and chemical substances-induced epilepsy models in mice. Furthermore, the regulatory effect of GEAT decoction on seizure severity, cognitive function, inflammation, and oxidative stress in PTZ-kindling mice was also assessed to support the anticonvulsant properties attributed to the two interactions herbs in traditional clinical practice.

Preparation of GEAT decoction
GEAT decoction in our study was composed of G. elata ("Tianma" in Chinese) and A.tatarinowii ("Shichangpu" in Chinese). Herbs were purchased from Beijing Tongrentang pharmaceutical chain Co., Ltd. Briefly, G. elata (30 g) and A.tatarinowii (15 g) were soaked in 500 mL of distilled water under normal temperature for 60 min before being boiled for 0.5 h. Filter and collect the filter liquor, and then add 250 mL of distilled water to the residue and continue to boil for 25 min. Afterward, combined the filter liquor and then concentrated using a rotary evaporator (model: Heidolph Hei-VAP). The concentrated solution was transferred to a glass bottle and then reserved at 4 °C in the ice box.

UHPLC-MS/MS analysis of GEAT decoction
UHPLC-MS/MS (Thermo Fisher Scientific, USA) equipped with an electrospray ionization (ESI) source was applied for the qualitative analysis of phytochemical compounds from GEAT decoction.

Animals
SPF adult male Kunming mice (Scxk (Guangdong) 2020-0051) weighing between 24 and 28 g were obtained from the BesTest Bio-Tech Co., Ltd. They were housed in the regulated environment (23 ± 2 ℃; 50 ± 10% humidity, 12 h light/dark cycle) with free access to pellet food and water. All experiments complied following the guidance of management regulations of Guangdong Medical Laboratory Animal Center (Guangdong, China), and were carried out by the NIH guidelines. All experimental protocols were approved by the Animal Care Committee of Zunyi Medical University (Zhuhai, China) (ZYLS- [2020] No. 2-081).

Acute seizures test
The mice were divided randomly into five groups, with 12 mice in each group. The model control mice received 0.9% sodium chloride (NaCl) containing 0.5% Poloxamer. The mice of the positive control group received CBZ (a most commonly used antiepileptic drug), at a dose of 50 mg/kg. The mice of the treated groups received three different doses of GEAT decoction at 50, 100, and 200 mg/kg, respectively. The different doses of GEAT decoction, normal saline, and positive drugs were treated to mice in a double-blind way, and the mice were orally administrated doses of NaCl, CBZ, or GEAT decoction once a day for consecutive 7 days.

Chronic seizures test
The mice were divided randomly into 6 groups, 12 mice in each group. The different doses of GEAT decoction, normal saline, and positive drugs treated in mice in a double-blind way, and the mice were orally administrated test doses of NaCl, CBZ, or GEAT decoction once a day for consecutive 28 days. The mice in the control group received 0.9% sodium chloride (NaCl) containing 0.5% Poloxamer. Except for the mice in the normal control group, all the mice in the other group were administered PTZ in a dose of 35 mg/kg for 14 days on alternate days. The mice in the control group received normal saline injections. The timeline of the experimental procedure is shown in Fig. 1.

MES test
MES tests were carried out according to the previously described method Krall et al. 1978). Thirty mice were randomly divided into five groups and administered with the double-blind method as mentioned earlier. 0.5, 1, 2, and 4 h after the last administration, mice were stimulated with a 0.25 s, 64 Hz, 50 mA stimulus by ear-clip electrodes using an electronic generator (Rodent Shocker). Mice were considered "protected" when full hind-limb tonic extension (HLTE) was absent from them Goerl et al. 2021). The number of mice protected from HLTE induced by electrical stimulation was recorded after the last drug was administered 0.5, 1, 2, and 4 h.

Acute PTZ-induced mouse seizure model
Mice from each group treated the drug doses described in the experimental groups (0.9% NaCl, CBZ 50 mg/kg, GEAT decoction 50 mg/kg, GEAT decoction 100 mg/kg, or GEAT decoction 200 mg/kg), for 7 days. One hour after the last dose, 85 mg/kg of freshly prepared solution of PTZ was administered subcutaneously to all the mice. Then, the tested mice were placed immediately in a transparent plastic square box for observation for 20 min. Mice were considered "protected" when there is the absence of a single 5-s episode of clonic spasms (Krall et al. 1978). Latent time for the onset, the number of animals of generalized tonic-clonic seizures (GTCS), clonic seizures (CS) as well as mortality were recorded for 20 min after PTZ injection. In addition, seizure severity was evaluated primarily based on the Racine scale with minor modifications. Briefly, stage 0: no response; 1: facial and ears twitching; stage 2: hyperactivity, vibrissae twitching, and myoclonic jerks; stage 3: unilateral forelimb clonus; stage 4: clonic convulsions with preservation of righting reflex; stage 5: generalized GTCS loss of postural control (Zhang et al. 2019).

Chronic PTZ-induced kindling mice model
The mice were randomly divided into six groups: normal group, in which each mouse was daily oral administration of NaCl; Model group (NaCl + PTZ), in which each mouse was daily oral administration of NaCl 30 min before administered a sub convulsive dose of PTZ (25 mg/kg); CBZ + PTZ group, in which each mouse was daily treated with CBZ (50 mg/ kg) 30 min before PTZ injection; GEAT decoction (50, 100 and 200 mg/kg) + PTZ group, in which each mouse was daily treated with a corresponding dose of GEAT decoction 30 min before PTZ injection. All groups were treated for 28 days. The Racine Scale was used to record and assess the seizure severity of mice within 20 min after each PTZ injection (Zhang et al. 2019). 24 h after completion of the kindling test, the behavioral assessment models were carried out to evaluate the ameliorative effects of GEAT decoction on anxiety, and cognitive function in the kindled mice. After completion of the behavioral testing, all mice were immediately executed. Blood from the heart was collected and centrifuged at 1000 g for 5 min and collected plasma for standby. The brain tissue was removed and the hippocampal was collected and immediately stored at − 20 ºC. The proinflammatory cytokines TNF-α, IL-1β, and IL-6 levels were tested using enzyme-linked immunosorbent assay (ELISA, Elabscience Biotechnology Co., Ltd) with the sensitivity of 18.75 pg/mL, 4.688 pg/mL, and 9.375 pg/mL, respectively. In addition, the biomarkers of oxidative stress including SOD, MDA, GSH, and CAT content in the hippocampus was also detected using corresponding assays (Nanjing Jiancheng Reagent Co., Ltd). The performance of the biochemical tests strictly followed the instructions of each assay. Besides, all of the mice in the study underwent a battery of behavioral tests, in the following order: high plus maze (29 th day after the induction of status seizure) and open field test (30 th day after the induction of status seizure).

3-MP-induced seizures test
3-MP-induced seizures tests were carried out according to previously described methods Bai et al. 2019). Mice grouping and treatment in this test were similar to that of the PTZ-induced acute seizure test. 30 min after the last treatment, 60 mg/kg of freshly prepared solution of 3-MP was administered subcutaneously to all the mice. The latency to myoclonic jerks was noted along with the occurrence of generalized tonic seizures and CS. The mice's death was also monitored. The observation period was 20 min for an individual mouse.

Elevated plus maze test
The elevated plus maze (EPM) test is a simple method to assess anxiety-like behaviors in mice by estimating contradictory and conflicting behavior between the exploring characteristics of animals to new/different environments and the fear of hanging open arms forms (Guillén-Ruiz et al. 2021). The maze (Shanghai xinruan Information Technology Co., Ltd, XR-XG201) consists of a plus-shaped platform 50 cm above the floor with two open (35 cm long × 5 cm wide) arms, a central square (5 cm long × 10 wide), and two closed (35 cm long × 5 cm wide × 15 cm height) arms. Based on a standard type preexperiment, the high plus maze test was performed on 29 th day when PTZ was administered 24 h later to mice in PTZinduced chronic seizure model. Each mouse was placed in the central area of the maze and monitored for 10 min, and the times spent and residence time of mice entering the open arm within 10 min was recorded by software monitored during the test.

Open field test
The open field test (OFT) was mainly and commonly used to observe the locomotor activity, exploratory behavior, and neuropsychiatric changes of experimental animals in new and different environments (Flores-Fuentes et al. 2021). The opening box inner with the floor divided into 9 equal quadrants (Shanghai, XR-XZ301) is 50 cm in diameter and 40 cm in height. Based on a standardtype pre-experiment, the OFT was performed on the 30th day when PTZ was administered 48 h later to mice in the PTZ-induced chronic seizure model. The mice were placed in the opening box inner, and the video analysis system was used to analyze the total distance and time spent on mice entries into the central zone within 5 min.

Statistical analysis
Data in this study were presented as mean ± SEM. Oneway analysis of variance (ANOVA) followed by the Bonferroni post hoc test was performed to analyze the data. Kruskal-Wallis ANOVA was applied for Racine score. The Chi-square test was used for counting data. Values of P < 0.05 were considered statistically significant. The statistical analyses were conducted using GraphPad Prism 7 software.

Chemical analysis of the GEAT decoction
The UHPLC-MS/MS technology was carried out for the preliminary analysis of GEAT decoction. The total ion chromatogram (TIC) was extracted as demonstrated in Fig. 2. A total of 173 components were identified from GEAT decoction using a broad targeted metabolomics approach based on UHPLC-MS/MS. Among them, the main structural types are benzene and substituted derivatives, carboxylic acids and derivatives, cinnamic acids and derivatives, furanoid lignans, prenol lipids, phenol esters, pyridines and derivatives, Fatty Acyls, etc. A total of 20 components were more than 1% relative, as shown in Table 1.

Effect of GEAT decoction on MES-induced seizures
The evaluation of the effects of GEAT decoction on the MES test in mice was shown in Table 2. As can be seen from Table 2, oralally administration of GEAT decoction for 14 days dose and time-dependently protected mice from hind-limb tonic extension (HLTE) in comparison with the control group. Specifically, 1 h after the last drug administration, GEAT decoction at a dose of 50, 100, and 200 mg/ kg protected mice from HLTE to 33.3, 66.6, and 83.3% respectively, while 50 mg/kg CBZ used as a reference drug also showed 83.3% protection in MES model of seizures as compared to the control group. The projected number of animals reduced in 4 h after the last drug administration, which may be related to the metabolism and excretion of active ingredients.

Effect of GEAT decoction on PTZ-induced seizures
In PTZ induced acute seizure model, compared to the model group, GEAT decoction exhibited a significant delay in the latency of seizures at the tested dose of 100 and 200 mg/kg with mean seizure thresholds of 243.5 and 254.5 s, respectively (Table 3). In addition, GEAT decoction at 100 and 200 mg/kg offered 50.0, 66.7, and 83.3, 66.7% protection against PTZ-induced GTCS and mortality, while CBZ at 50 mg/kg produced a slightly smaller proportion of protective activity as GEAT decoction at 200 mg/kg (Table 3). In contrast, the GEAT decoction in all experimental groups did not completely inhibit clonic seizures. However, compared to the saline-treated control group, seizure scores in GEAT decoction decreased in a dose-dependent manner, whereas mice in the model group showed significantly higher seizure scores after administration of 85 mg/kg of PTZ, showing a good antiepileptic effect. Similar, in PTZ induced chronic seizure model, as shown in Fig. 3, injection of PTZ to mice resulted in degrees of seizure severity and resulted in more complex seizures, while treatment with GEAT decoction dosedependently produced retardation in the seizure scores for all the treatment days.

Effect of GEAT decoction on 3-MP-induced seizures
As shown in Table 4, with regard to the latency to tonic seizures, an obvious decrease in the NaCl group was observed.
Oral administration of GEAT decoction resulted in different degrees of extension on onset latency. Especially, GEAT decoction at 200 mg/kg significantly inhibited and delayed the onset of myoclonic seizures with a seizure threshold  (Table 4). Taken together, GEAT decoction reduced the severity of convulsive activity and also prevent tonic-clonic seizures in the 3-MPinduced drug-resistant seizures test.

Effects of GEAT decoction on oxidative stress parameters
PTZ-induced kindling markedly elevated oxidative stress in the mice. In terms of quantification of oxidative stress parameters in the hippocampus, all treatments presented the higher activity of SOD when compared to the model (saline, PTZ existence) group. Particularly, pre-treatment GEAT decoction at 200 mg/kg significantly elevated SOD activity in the hippocampus compared to the model group (P < 0.01) (Fig. 5A). About the CAT, a significant decrease in the model group was observed when compared to the normal group (P < 0.01). Administration of GEAT decoction at the dose of 200 mg/kg produced a better elevation effect of CAT activity in the hippocampal of mice in comparison with the model group (P < 0.01) (Fig. 5B). In addition, results showed an enhanced production of MDA as well as a reduced production of GSH in hippocampal in PTZ induced mice. Interestingly, these changes were reversed upon GEAT decoction treatment in varying degrees ( Fig. 5C and  D). However, the levels of these oxidative stress parameters in animals treated with CBZ were not significant changed in comparison with the model group (P > 0.05).

Effect of GEAT decoction on cognitive and behavioral functions in the EPM test
It has been proposed that depression and anxiety symptoms are a frequent occurrence in epilepsy, therefore anxiety-like behavior was evaluated in this study. As shown in Fig. 6A   Fig. 3 Effect of GEAT decoction and CBZ on subcutaneous PTZ-kindling seizure in mice for 14 injection every two days.
The behavioral seizure and severity scale was observed and evaluated using the Racine scale as indicated earlier in PTZ-induced acute seizure test. Data expressed as Mean ± SEM, n = 12 mouse per group. Statistical analyses were implemented using one-way ANOVA test. * P < 0.05, ** P < 0.01, *** P < 0.001 compared with saline group on the same day

Effect of GEAT decoction on cognitive and behavioral functions in OPT
Likewise, the time spent and distance in the central areas were used as an anxiety-like indicator determined in OFT. As shown in Fig. 6D, the time spent in the central area for mice in the normal group was 34.0 ± 2.7 s and 15.3 ± 3.6 s for mice in the model group. For GEAT decoction at doses of 50, 100, and 200 mg/kg, the time spent in the central area was 20.0 ± 3.9, 25.8 ± 3.3, and 34.3 ± 8.9 s, respectively. As shown in Fig. 6E, GEAT decoction at doses of 100 and 200 mg/kg significantly increased total distance of the mice moving in the central are compared that of mice in model group. However, changes in these indicators in CBZ and GEAT decoction (50 mg/kg) treatment did not show significant differences in comparison with the model group in the same period of treatment, as shown in Fig. 6D and E. The behavior trace of GEAT decoction on subcutaneous PTZ-induced kindling mice in OPT are shown in Fig. 6F.

Discussion
Epilepsy induced by many reasons is the most common chronic brain disease, affecting about 70 million people worldwide (Thijs et al. 2019). Traditional Chinese herbal medicine has a long history of use for treating epilepsy. Currently, herbal treatments for seizures has attracted lots of attention globally. In addition, the herbal treatment appears to be inexpensive, safe, easy to get, and effective in treating epilepsy . So far, more than 14 kinds of TCM prescriptions or preparations for the treatment of various epilepsy, especially intractable epilepsy has included in the 2020 edition of Chinese Pharmacopoeia Mean ± SEM. Statistical analyses were implemented using one-way ANOVA test. * P < 0.05, ** P < 0.01, *** P < 0.01 compared with model (saline, PTZ existence) group. # P < 0.05, ## P < 0.01, ### P < 0.001 compared with normal (saline, PTZ absence) group (Chinese Pharmacopoeia Committee 2020). According to statistics and analysis, the commonly used and clinically effective drug pairs "G. elata-A. tatarinowii" are the most representative clinically valuable drug pair in the treatment of epilepsy and seizures in folk medicine in China (Bao et al. 2012;Zhao et al. 2018;Bai et al. 2019). There is no doubt that the effectiveness of the compatibility of these classic drug pairs has been verified in clinical practice for a long time, but modern systematic pharmacological evaluation and mechanism research are relatively lacking. Therefore, in this study, three classical animal models of epilepsy were performed to evaluate the antiepileptic effect and related mechanism of GEAT decoction. Additionally, the EPM test and OPT were performed to examine the impact of GEAT decoction on the cognitive and behavioral functions of PTZ-kindling mice.
In this study, UHPLC-MS/MS was first utilized to identify the chemical compounds of GEAT decoction. In total, 173 compounds were identified from GEAT decoction, and 20 of them were more than 1% relative. Among them, researchers demonstrated that some potential compounds in GEAT decoction, such as α-asarone, gastrodin, and parishin C, etc., showed anticonvulsant efficacy by decreasing the seizures . Then, we evaluated the anticonvulsant effects of GEAT decoction at different dosages on three different acute seizure models, the MES, 3-MP, and PTZ tests. The results demonstrated that mice treated with GEAT decoction (50, 100, 200 mg/kg, po.) delayed the onset of myoclonic seizures, inhibited generalized seizures in the MES, PTZ and 3-MP induced seizure models. Especially, GEAT decoction at 200 mg/kg delayed the onset latency and prevented the severity of PTZ-induced seizures, indicating Effect of GEAT decoction and CBZ on levels of main oxidative stress markers in the hippocampus of subcutaneous PTZ-kindling mice. A, SOD activity; B, CAT activity; C, MDA levels; D, GSH levels. Data presented as Mean ± SEM. Statistical analyses were imple-mented using one-way ANOVA test. * P < 0.05, ** P < 0.01 compared with model (saline, PTZ existence) group. # P < 0.05, ## P < 0.01, compared with normal (saline, PTZ absence) group its good anticonvulsant effect. In addition, similar dosages of GEAT decoction also performed well in MES and 3-MP seizure models. Therefore, this study provides proof of concept that GEAT decoction are pharmacologically active in vivo with a dose-dependent manner, which possessed a therapeutic potential to prevent and control seizures. It is worth noting that 3-MP is an experimental model of drugresistant seizures associated with P-glycoprotein (Pgp) overexpression (Pérez-Pérez et al. 2021), further studies are essential to determine if GEAT decoction is effective in more experimental models of drug-resistant epilepsy. Moreover, the repetitive administration of 3-MP induced seizure test should be established for determination the Pgp expression and/or function of the cortex and hippocampus in GEAT decoction-treated mice to explore the synergistic effect of GEAT decoction combination with currently available AEDs.
Evidence suggests that inflammation strengthens excitability of neuronal, and consequently prolongation of seizures and initiation of cognitive dysfunctions, while alleviation of inflammation displayed anticonvulsant effects in intractable epilepsy (Kaur et al. 2015). Inflammatory mediators induced by cytokines may be not only a complication of epilepsy, but also an internal inducement of some epilepsy diseases. For example, high levels of inflammatory mediators, including IL-1β, IL-6, and TNF-α were detected in the brain tissue of patients with intractable epilepsy (temporal lobe epilepsy caused by cortical dysplasia) (Bauer et al. 2017;Elgarhi et al. 2020;de Lima Rosa et al. 2021). In our study, we found that PTZ induced generalized seizures and elevated IL-1β, IL-6, and TNF-α levels in kindled mice blood and brain. Gratifying, in this study the administration of GEAT decoction dependently reversed the increase of inflammatory cytokines IL-1β, IL-6, and TNF-α levels in the serum and brain tissues of PTZ-kindling mice. Therefore, GEAT decoction may have potential value in the management of inflammatory diseases accompanied by epilepsy.
Studies have found that oxidative stress and mitochondrial dysfunction may be the causes and the results of genetic and acquired epilepsies (Chindo et al. 2021). Increased production of free radicals produces unwanted side or harmful effects on the structure and functions of neurons, changing or damaging the biological function regulation of the central nervous system. In particular, the increase in the synthesis and release of reactive oxygen species lead to great damage to the steady-state of the oxidation potential of the central nervous system (Frantz et al. 2021). Thus, removing excessive hydroxyl radical, peroxy radical, and superoxide radical, as well as elevating the activity of superoxide dismutase and glutathione peroxidase are very beneficial to ease symptoms or to control seizures. In the pathogenesis distance travelled in the central areas in OPT; F, Behavior trace of mice in OPT. Statistical analyses were implemented using one-way ANOVA test and Chi square test. * P < 0.05, ** P < 0.01 compared with model group (saline, PTZ existence). # P < 0.05, ## P < 0.01, compared with normal group (saline, PTZ absence) of chronic epilepsy, a large great number of superoxide anions free radicals were produced, while the endogenous antioxidant enzymes SOD, GSH, GSR, and CAT are rapidly consumed, resulting in the excessive production of toxic lipid peroxide that then led to oxidative stress and neuronal death. In addition, in PTZ-induced kindling in mice, it was found that reactive oxygen species were activated, and its production agrees with a decrease in antioxidant-related enzymes (Frantz et al. 2017;Chindo et al. 2021). In this study, we found that mice treated with GEAT decoction displayed a dose-dependent reduction in the production of MDA in PTZ-kindled mouse hippocampus, while showing an increase in activities of CAT and SOD activities, as well as exhibited an increase in the production of GSH when compared with that of PTZ-kindled epileptic mouse models. In other words, GEAT decoction improved the antioxidant capacity of brain tissue, and reduced lipid peroxidation and peroxidation damage in the mouse brain, thus corroborating the therapeutic benefits of GEAT decoction in the management of epilepsy.
It has been proposed that cognitive impairment, anxiety and depression are common accompaniments neurological of chronic epilepsy (Chindo et al. 2021). Patients with longterm seizures can cause diversified degrees of brain injury and abnormal emotions during seizures (Sharma et al. 2021). More seriously, most cognitive impairment occurs after recurrent seizures or status epileptics, and the frequency, duration, and severity of seizures are closely associated with the severity of cognitive impairment (Shuman et al. 2020). The EPM test and OPT are some of the most widely used tests to assess depression/anxiety and cognitive dysfunction in animals (Knight et al. 2021). Thus, in our study, we explored the effects of GEAT decoction on anxiety and cognitive dysfunction in the PTZ-kindled epileptic mouse model using OFT and EPM tests. Data have shown that the time spent in the central areas of OFT and in the open arms of EPM was decreased in PTZ-induced mice, which means a state of avoiding fear and anxiety behavior in these kindling mice. Whereas, the GEAT decoction treatment mice spent more time on the open arms of the EPM test and made more open arms entries than non-GEAT decoction-treated mice. Similarly, GEAT decoction also spent more time in the center zone of the OPT, made more center zone entries and traveled a greater distance in center zone than controls. The results preliminary demonstrated that GEAT decoction evidently improved anxiety-like behavior and cognitive impairment in PTZ-kindled epileptic mouse, which supported the traditional records that the couplet medicinal of G. elata and A. tatarinowii relieving convulsion and stress. However, no doubt that GEAT decoction capable to reduce anxiety and stress in this study, more in-depth studies on the alleviation of mental stress of GEAT decoction in various aspects are needed.

Conclusion
GEAT decoction showed outstanding protected activities in MES, PTZ, and 3-MP induced models of seizures. Especially, GEAT decoction has a promising activity in reducing inflammation and oxidative stress, as well as improving anxiety behavior in PTZ-kindled mice, confirming the potential efficacy of GEAT decoction in the prevention and treatment of epilepsy. Thus, GEAT decoction can be used to inhibit neuroinflammation, suppress oxidative damage and prevent cognitive deficits in chronic epilepsy mice. Further experimental and clinical studies could provide deep insight into the best compatibility proportion, clinical effect, and mechanistic pathway involved in the management of epileptic seizures by GEAT decoction.