A Study to Investigate Anticonvulsant Activity of Hydroalcoholic Extract of Brassica Alba Seeds in Experimental Animal Models

Epilepsy is a neurological disorder of the brain which is characterized by repeated seizures. Here seizures refer to temporary symptoms such as abnormal movement of the body (repeated jerks and stiffness in arms and legs) against their will. In epilepsy, there is a surge of abnormal and excessive neuronal firing in the neurons of the brain which leads to the development of seizures. During a seizure all the neurons in the brain fire at the same time at around 500 times per second which is much faster compared to normal neuronal action potential (5-50 times). And this abnormal neuronal firing and action potential is mainly due to the imbalance between inhibitory (GABA) and excitatory (Glutamate) neurotransmitters.^[1]

A systematic review indicated that the incidence of epileptic seizures is significantly higher in low- and middle-income countries (LMIC) than in high-income countries (HIC). In LMIC, epileptic seizures account for approximately 1% of hospital admissions and 3% of Emergency Department (ED) visits. This elevated incidence is associated with increased rates of physical and psychiatric comorbidities, greater healthcare utilization, and higher mortality rates compared to the general population. ^[2,3] Plants are considered an alternative or complementary form of medicine for treating seizures. They can be used in the form of herbal decoctions, acupuncture, infusions, and powders. According to the 2020 Committee of National Pharmacopeia more than 20 traditional Chinese patent medicines are approved for therapeutic use. Despite the development of around 15 third-generation AEDs in the past thirty years, these treatments are not effective for 20-30% of patients. Furthermore, over 40% of patients report pharmacoresistance to current doses, and exclusive reliance on Western medicine can lead to adverse effects, addressing only symptoms rather than the underlying causes of epilepsy. Consequently, there is a pressing need to explore novel drugs derived from herbal sources to overcome these limitations and develop medications with different mechanisms of action and minimal pharmacokinetic interactions.^[4]

Mustard is a group of dried and mature seeds of Brassicaceae species. The plant of white or yellow mustard seeds is known by different scientific names such as Brassica alba, Brassica hirta, and Sinapis alba. This group of species is known for its pungent, bitter, sharp, unctuous, and hot-flavored seeds that can be used commercially as condiments. The white mustard plant is a winter-spring plant.^[5] The seeds include crystalline primary bioactive substances called sinalbin, mucilage, lecithin, myrosin, 4% ash (potassium, calcium, and magnesium phosphates), and a bland fixed oil that makes up roughly 23–25% of the seeds. They also include water, proteins, riboflavin, thiamine (B1), and vitamin A. White mustard seeds are found to have a variety of chemical constituents. They are reported for the presence of essential oils, fatty oils (glycerides of oleic acid, linoleic, linolenic, palmitic, stearic, arachidic, erucic), phytosterols, polysaccharides and also found to have large amount phenolic compounds, carbohydrates, antioxidants and a smaller amount of flavonoids.^[6,7] The experimental studies done on different parts of the white mustard plant suggests antiproliferative, proapoptotic, antibacterial, antioxidant, nephroprotective, anti-inflammatory, ant analgesic, anticancer and immunomodulatory properties.^[8]

Traditionally the White mustard seeds have been used in the treatment of diseases such as epilepsy, and hysteria. Edible oil from the White mustard seeds is applied locally in case of headache, cerebral congestion, or chest pain as a mustard bath. Yellow mustard calms vata and kapha and is beneficial for ailments related to the head and ear. It can be applied in the form of liniment to swollen joints. The seeds are used as stimulant, emetic, expectorant, appetizer and diuretic. They are also used in the treatment of amenorrhoea and dysmenorrhoea as mustard hip bath. The tea made of seeds is used in treating sore throat and relieves breathing difficulty. The flowers when made into a paste with water helps in treating epigastrium in vomiting, in the treatment of pertussis, help in difficulty in breathing, cholera. The same paste can be applied in the calf of the leg in case of muscle cramps, delirium. A foot bath of mustard seeds can be employed in extreme fever.^[9]

In traditional Chinese medicine, white mustard seeds are utilized in the form of Centipede Scorpion pills, which consist of a combination of nine active compounds, with Brassica alba (the seeds) being one of them. This formulation is recognized for its application in the treatment of seizures. Furthermore, the oil extracted from white mustard seeds has diverse applications, including cooking, food preservation, body and hair care, and as a feedstock for biodiesel. It is also used as a diesel fuel additive and as an alternative biofuel. However, scientific evidence regarding the antiepileptic properties of white mustard seeds is lacking. Therefore, this study aims to assess the antiepileptic efficacy of white mustard seeds and confirm their traditional use as an antiepileptic agent.^[10,11]

MATERIALS AND METHODS:

Chemicals such as Diazepam, Pentylentetrazole, and phenytoin of pure analytical grade were procured from E. Merck (India) Ltd, Mumbai and all other chemicals and reagents of pure analytical grade were procured from local suppliers. Experiment was performed as described in the standard bibliography, literatures and textbooks.

Plant collection, authentication and extract preparation

The seeds of Brassica alba were collected from the local market of Mangalore, Dakshina Kannada in May 2024 and verified by Dr. Siddaraju M.N, Ph.D., Department of Botany, University College of Mangalore. Around 150g of the powdered sample of the seeds was extracted by maceration with ethanol and distilled water in a ratio of 1:1 followed by occasional stirring. This mixture was allowed to stand at room temperature for at least 3-7 days. After 72 hours, the mixture was filtered, the residue was re-macerated twice for the same hours, and then the mixture was filtered again. The combined filtrate was then dried and the residue obtained was discarded. The dried extract later stored in a refrigerator until further use.^[12]

Experimental animals

Swiss Albino mice (25-30g) of either sex were used for this study. They were maintained under standard conditions (temperature 22±2°C, relative humidity 60±5% and 12 h light/dark cycle) and being given free access to standard pellet diet and water ad libitum. The animals were be housed in a sanitized environment in polypropylene cages containing sterile paddy husk as bedding. The Institutional Animal Ethics Committee reviewed and approved the experimental protocol (Approval no: SCP/IAEC/F150/P209/2023). All the procedures were performed in accordance with Institutional Animal ethics committee constituted as per the direction of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CCSEA).

Phytochemical Screening

The hydroalcoholic extract of Brassica alba seeds was qualitatively investigated for the presence of different phytochemicals according to the standard procedures

Experimental Design

Maximal Electroshock Induced Convulsion In Mice

The Maximal electroshock induced convulsion model identifies drugs that effectively treat grand mal epilepsy. The Swiss albino mice (25-30g) of either sex will be divided into four groups. Each group consisted of six animals each. For this experiment the mice received 200 mg/kg  and 400 mg/kg of hydroalcoholic extract of Brassica alba seeds for a period of 14 days. On the day of experiment the control group received vehicle and standard group received Phenytoin 25mg/kg intraperitoneally. 30 mins after the treatment with standard drug and 60 mins after the treatment with extracts and vehicle, all the groups will be induced with MES-convulsions resembling the generalized tonic-clonic seizure/ Grandmal epilepsy by the application of electrical stimulus that carries current of 30mA, 50Hz for a duration of 0.2 Sec. After inducing seizures using MES, the animals were closely monitored for 2 min.The suppression of hind limb tonic extension (HLTE) was taken as measure of efficacy in this test. Percent of inhibition of seizures relative to the controls was calculated.^[13]

Pentylenetetrazole Induced Convulsions

Pentylenetetrazole is a tetrazole derivative. This chemical can cause convulsions in several animal species by interfering with the inhibitory GABAergic neurotransmission. The PTZ test screens medicines for their effectiveness in treating petitmal epilepsy or absent seizures. For this experiment the mice were divided into 4 groups. Each group consisted of 6 animals each. For this experiment the animals were treated with the extract for a period of 14 days. On the 14^th day the animals received 200, and 400?mg/kg doses, vehicle through the oral route and diazepam 4mg/kg intraperitoneally. 30 mins after the treatment with standard drug and 45 mins after the treatment with vehicle and extracts, the groups were induced with clonic-type convulsions by intraperitoneal administration of PTZ (80mg/kg). After administration of PTZ, the animal was observed closely for onset of action. The percentage protection against morality, onset time of the convulsions and duration of convulsions were measured. Reduction in the duration of convulsion was considered as the parameter for the evaluation and analyzed with that of the control treated with vehicle.^[14]

RESULTS

The preliminary phytochemical screening of the hydroalcoholic extract of Brassica alba seeds revealed the presence of Flavonoids, Phenols, Steroids, Carbohydrates, Volatile oil, Proteins and Saponins.

Maximal electroshock induced convulsion

The study found that the hydroalcoholic extract of Brassica alba seeds exhibits significant antiepileptic activity in a dose-dependent manner against convulsions induced by maximum electroshock (MES) in mice. In the control group, notable durations of convulsions were recorded without inhibition. In contrast, Phenytoin at 25 mg/kg offered complete protection. The extract at 200 mg/kg provided 34.0% protection, while 400 mg/kg resulted in 50.65% protection. The effectiveness was measured by the suppression of hind limb tonic extension (HLTE), with higher doses yielding greater protection.

The onset and duration of convulsions in the PTZ model were assessed for two test groups (200 mg/kg and 400 mg/kg) and compared to a standard group (Diazepam at 4 mg/kg) and a control group. Both test groups showed a significant reduction in convulsion duration, with the 400 mg/kg dose exhibiting superior effects over the 200 mg/kg dose. The control group had a mortality rate of 66.66%. The seed extract at 200 mg/kg achieved 50% inhibition of convulsions, while the 400 mg/kg dose resulted in 33.33% inhibition.