Evaluation of the Anticonvulsant Activities of Gastrodia Elata Bl.- Acorus Tatarinowii Decoction on Experimentally Induced Seizures in Mice

Epilepsy is a serious public health problem in the world. At present, the effect of drug treatment of epilepsy is are not satisfactory. Medicinal plants as pharmaceuticals and for healthcare treatments in the management of epilepsy in China for many centuries. Especially, Gastrodia elata Bl.-Acorus tatarinowii, as a classic and important herb pairs in folk medicine, has been used in folk medicine to control seizures. However, the animal experiment data of its anticonvulsant effect is limited in the literature. The objective of this study was to mainly analyze the anticonvulsant activity of Gastrodia elata-Acorus tatarinowii (GEAT) decoction in maximal electroshock (MES), pentylenetetrazole (PTZ) and trimercaptopropionic acid (3-MP) induced seizures in mice, providing scientic basis for the treatment of convulsive disorders in traditional medicine. In addition, the improvement effect were examined on seizure severity, anxiety, cognitive dysfunction, inammation and oxidative stress in PTZ kindled mice. The results showed that GEAT decoction dose-dependently protected mice against MES, 3-MP and PTZ induced acute seizures. Meanwhile, GEAT decoction ameliorated seizure severity, decreased the accumulation of inammatory mediators TNF-α, IL-1β, and IL-6, mitigated oxidative stress, as well as alleviated anxious-like behavior and cognitive decits in PTZ-kindled mice. Our data evidenced that the anticonvulsant properties attributed to GEAT decoction as adjunctive therapy for epileptic patients in folk medicine.


Introduction
Epilepsy is a common and complex neurological disease, affecting more than 65 million people around the world. There are more than 12 million epilepsy patients in China, of which about 8 million are active epilepsy patients, and about 40, 0000 new epilepsy patients are added every year Johnson, 2019). Epilepsy has become the second largest neurological disease in China, which is second only to headache, especially most of epileptics are children and elderly patients (China Association against Epilepsy, 2015). In addition, at this stage, China has entered population-aging society and accompanied by three-child policy fully implemented, so that the number of epileptics in China will increase for a considerable period of time in the future. Compared with elderly patients with epilepsy, the pathogenesis of epilepsy in childhood are more complex and diverse. More importantly, a considerable part of them belong to intractable epilepsy, which produces a great impact on the cognitive mental, psychological and social functions of childhood with epilepsy (Hwang et al. 2019). Due to the poorly understanding of the pathogenesis to the lack of signi cant therapeutic regimens, the treatment of epilepsy, especially intractable epilepsy, with low cure rate, lack of treatment methods and scarce drugs has become a di cult problem in the medical eld (Löscher and Klein, 2020). Therefore, the research on epilepsy and its treatment has extremely important practical signi cance and urgency.
At present, the major choice for the treatment of epilepsy in the clinic is still mainly drugs (He et al. 2018). However, the existing clinical antiepileptic drugs (AEDs) are only about 2/3 of epileptic patients achieve a satisfactory seizure control, and the treatment effect is poor or ineffective for the other 1/3 of epileptic patients (He et al. 2018; 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, although chemical drugs are widely used because of their convenient use, rapid action, high clinical e cacy and good availability, they have side effects due to the need for long-term use, which will damage your health (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 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).
Traditional Chinese medicine has a long history in the treatment of epilepsy, which was recorded in the classical masterpieces Inner Canon of Huangdi ( ) 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 seizure in children (Bai et al. 2019

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. Brie y, 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 lter liquor, and then add 250 mL of distilled water to the residue and continue to boil for 25 min.
Afterwards, combined the lter liquor and then concentrated using a rotary evaporator (model: Heidolph Hei-VAP). The concentrated solution was transfer to a glass bottle, and then reserved at 4 °C in ice box.

Treatment processes
The mice were divided randomly into 6 groups, 6 mice in each group. The normal control and model control mice received 0.9 % sodium chloride (NaCl) containing 0.5% Poloxamer. The mice of positive control group received CBZ (a most commonly used antiepileptic drugs), at a dose of 50 mg/kg. 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 100 mg/kg), for 14 days. After the last dose of the drugs, 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 was considered "protected" when the duration of generalized tonic seizure was less than 5 s. Latent time for the onset, the number of animals of tonic and clonic seizures as well as the mortality were recorded for 20 min after PTZ injection.

PTZ-induced chronic seizure 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 subconvulsive dose of PTZ (25 mg/kg); CBZ+PTZ group, in which each mouse were 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 were daily treated with corresponding dose of GEAT decoction 30 min before PTZ injection. All groups were treated for 14 days. The Racine Scale was used to recorded and assess seizure severity of mice within 20 min after PTZ injection. After the last drug administration, except for the normal control group, the mice were observed for 20 min and then 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 were removed and hippocampal was collected and immediately stored at −20 ºC. The pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 levels was tested using enzyme-linked immunosorbent assay (ELISA). In addition, the biomarkers of oxidative stress including SOD, MDA, GSH and CAT content in hippocampus was also detected using corresponding assays (Nanjing Jiancheng Reagent Co., Ltd). The performance of the biochemical tests was strictly follow the instructions of the each assay. Besides, all of the mice in the study underwent a battery of behavioral tests, in the following order: high plus maze (10 days after the induction of status seizure) and open eld test (10 days after the induction of status seizure). In this study, the OFT was performed at 10 days when PTZ was administered 2 h to mice in 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 movement time of mice in the central area within 5 min.

Statistical analysis
Data in this study were presented as mean ± SDE. One-way analysis of variance (ANOVA) followed by the Bonferroni post hoc test was performed to analyze the data, while Chi square test was used for counting data. Values of p < 0.05 were considered statistically signi cant. The statistical analyses were conducted using Prism 5 software.

MES test
The evaluation of the effects of GEAT decoction on MES test in mice was shown in Table 1. As can be seen from Table 1, the orally administration of GEAT decoction displayed signi cantly protected effect against convulsion caused by electrical stimulation in a dose-dependently manner at 50, 100, 200 mg/kg, which offered a maximum protections against generalized seizures (tonic hind limb extension) in 1h after the last drug administration. Speci cally, GEAT decoction at 50, 100 and 200 mg/kg provided 33.3, 66.6, 83.3 % protections against seizures in MES test, respectively. The protected number of animals reduced in 4 h after the last drug administration, which may be related to the metabolism and excretion of active ingredients.

PTZ-induced seizures
In PTZ induced acute seizure model, compared to the model group, GEAT decoction exhibited a signi cant delay in the latency of seizures at the tested dose of 100 and 200 mg/kg with a mean onset latency of 248.8 and 259.5 s, respectively (Fig.1A). In addition, GEAT decoction at 100 and 200 mg/kg offered 50.0, 75.0, and 83.3, 75.0% protection against PTZ-induced tonic seizure and mortality, while CBZ at 50 mg/kg produced same proportion of protective activity as GEAT decoction at 200 mg/kg (Fig.1B). In contrast, the GEAT decoction in all experimental groups did not develop clonic seizures. In PTZ induced chronic seizure model, as shown in Fig.2, injection of PTZ to mice resulted in degrees of seizure severity and resulted in more complex seizures, while treatment with GEAT decoction dose-dependently produced a retardation in the seizure scores for all the treatment days.

3-MP-induced seizures
As shown in Fig. 1C, regard to the latency to tonic seizures, an obvious decrease in in the NaCl group was observed.  (Fig.1D). Taken together, GEAT decoction reduced the severity of convulsive activity and also prevent seizures.

Effects of GEAT decoction on Pro-inflammatory cytokines
As shown in Fig.3, results showed elevated pro-inflammatory cytokines including IL-6, IL-1β and TNFα levels of in the model group as compared to normal group both in the hippocampus and serum of PTZinduced mice. In detail, in the hippocampus, administration of GEAT decoction at the dose of 50, 100 and 200 mg/kg produced a reduction of IL-6, IL-1β and TNF-α levels in different degrees ( Fig.1 A, B, C).
Especially, compared with model (saline, PTZ existence) group, treatment with GEAT decoction dramatically reversed the effect of PTZ on IL-6, IL-1β and TNF-α levels at the dose of 200 mg/kg (p<0.01).
Whereas, regarding IL-6 and TNF-α, treated GEAT decoction (100 mg/kg) and CBZ (50 mg/kg) showed lower levels than that of mice in model (saline, PTZ existence) group, but both of them did not demonstrate a protective effect regarding IL-1β when compared to the model group. In addition, a signi cant difference in IL-6, IL-1β and TNF-α levels between GEAT decoction 50 mg/kg groups and the PTZ group were not observed in our study. In the serum tests (Fig. 3 D, E, F), GEAT decoction at 50, 100 and 200 mg/kg produced a signi cant reduction in IL-1β level compared to the model (saline, PTZ existence) group. Changes in IL-6 and TNF-α levels are basically consistent with those in hippocampus. For the CBZ, no signi cant difference was observed among IL-6, IL-1β and TNF-α levels.

Effects of GEAT decoction on Oxidative Stress Parameters
In terms of quanti cation of oxidative stress parameters, in hippocampus, all treatments presented higher activity of SOD when compared to the model (saline, PTZ existence) group. Particularly, pretreatment GEAT decoction at 200 mg/kg signi cantly elevated SOD activity in the hippocampus compared to the model group (p<0.01) (Fig. 4A). Regard to the CAT, a signi cant 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 hippocampal of mice in comparison with model group (p<0.01) (Fig. 4B). In addition, results showed an enhanced production of MDA as well as a reduced production of GAH in hippocampal in PTZ induced mice. Interestingly, GEAT decoction treated reversed the changes in varying degrees (Fig. 4C, and D). However, the levels of these oxidative stress parameters in animals treated with CBZ was not signi cant changed in comparison with model group.

Elevated plus maze
It has been proposed that depression and anxiety symptoms are frequent occurrence in epilepsy, therefore anxiety-like behavior was evaluated in this study. As shown in Fig.5 A  There is no doubt that the effectiveness of the compatibility of this classic drug pairs has been veri ed in clinical practice for a long time, but modern systematic pharmacological evaluation and mechanism research are relatively lacking. Therefore, in this study, several classical animal models of epilepsy was performed to evaluate antiepileptic effect and related mechanism of GEAT decoction. Additionally, the elevated plus-maze and open eld tests were performed to examine the impact of GEAT decoction on anxiety-like behavior of PTZ-induced mice.
In this study, we rstly analyzed the anticonvulsant effects of GEAT decoction at different dosages on three different 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 an anticonvulsant activity 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 its good anticonvulsant effect. In addition, similar dosages of GEAT decoction also performed well in MES and 3-MP seizure models. Therefore, this study provide proof of concept that GEAT decoction are pharmacologically active in vivo with a dosedependent manner, which possessed a therapeutic potential to prevention and control seizures.
Evidence suggests that in ammation strengthen excitability of neuronal, and consequently prolongation of seizures and initiation cognitive dysfunctions, while alleviation of in ammation displayed anticonvulsant effects in intractable epilepsy . In ammatory mediators induced by cytokines may be not only a complication of epilepsy, but also an internal inducement of some epilepsy diseases. For example, a large number of in ammatory mediators, including IL-1β, IL-6 and TNF-α were detected in the brain tissue of patients with intractable epilepsy (temporal lobe epilepsy and epilepsy caused by cortical dysplasia) ( 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 in ammatory cytokines IL-1β, IL-6, and TNF-α levels in the serum and brain tissues of PTZ-induced seizures mice. Therefore, GEAT decoction may have potential value in the management of in ammatory diseases accompanied by epilepsy. Studies have found that epilepsy is also closely related to oxidative stress and mitochondrial dysfunction (Chindo et al. 2021). The production of free radicals plays an important role in biological function regulation, cell structure damage and the pathogenesis of neurodegenerative diseases of the central nervous system. In particular, the increase in the synthesis and release of reactive oxygen species is closely related to the oxidation potential of the central nervous system (Frantz et al. 2021). Thus, the scavenging of hydroxyl radical, peroxy radical, and superoxide radical, as well as stimulating the synthesis of superoxide dismutase and glutathione peroxidase are very bene cial to the treatment of epilepsy. In the pathogenesis of chronic epilepsy, a large number of superoxide anions can be produced, and the endogenous antioxidant enzymes SOD, GSH, GSR and cat are rapidly consumed, resulting in the production of a large number of toxic lipid peroxide increased and induced the oxidative stress. In addition, in PTZ-induced kindling in mice, it was found that reactive oxygen species was activated, and its production agrees with decrease in antioxidant related enzymes (Frantz et al. 2017;Chindo et al. 2021). In this study, we found that treated with GEAT decoction reduced MDA levels in PTZ-kindled mouse hippocampus, while increased CAT and SOD activities, as well as increased GSH levels when compared with that of PTZ-kindled mice. In other words, GEAT decoction improved the antioxidant capacity of brain tissue, reduced lipid peroxidation and peroxidation damage in mouse brain, thus corroborating the therapeutic bene ts of GEAT decoction in the management of epilepsy.
It has been proposed that cognitive impairment, anxiety and depression are common accompaniment neurological of chronic epilepsy (Chindo et al. 2021).    Effect of GEAT decoction and CBZ on subcutaneous PTZ-induced seizure in mice for 14 consecutive days. Racine Scale: 0, no response; 1, twitch of ears and face; 2, myoclonic seizures without rearing; 3, myoclonic seizures along with rearing; 4, rolling into one side with colic-tonic seizures; and 5, upside down along with generalized clonic-tonic seizures. Data expressed as Mean ± SEM, n=6 mouse per group.