Plant-based pharmacological alternatives in the seizure treatment: A patent review

Epilepsy, a neurologic condition associated with recurring seizures, affects around 70 million people worldwide and it is responsible for health costs. The current drug therapies used for treating epilepsy have low efficacy and many adverse side effects. In addition, drug resistance can also be found. The association of these factors considerably compromises the patient's pharmacotherapy care. Therefore, natural plant-derived products are promising therapeutic alternatives in the search for new curative options to treat epileptic seizures. Thus, the search was carried out at the patents database in the world, Espacenet, in January 2021 and compiled plant species (extracts, essential oils and isolated plant compounds) with a potential effect on the seizure, published between 2015 and 2020. In recent years, researchers and pharmaceutical companies have developed several methods for obtaining extracts, essential oils and purification of natural compounds, which have proven to be effective for the treatment of epileptic seizures in preclinical animal models. These findings hold potential promising for the treatment of epilepsy, especially for the management of drug-resistant, however, these reports are in their early stages and further studies need to be done for these compounds to be marketed.


Introduction
Epilepsy is a chronic neurological condition among the most prevalent central nervous system disorders, that affects approximately 70 million people worldwide (Beghi, 2020;Thijs et al., 2019;Thurman et al., 2011). It is characterized by the recurrence of unprovoked seizures caused by abnormal and excessive activity of brain neurons in an unpredictable manner (Fisher et al., 2005(Fisher et al., , 2017Stafstrom & Carmant, 2015). Epilepsy has no cure, but it can be treated using surgical procedures or medications (Byun et al., 2020;Catchpool et al., 2019). The first-line treatment involves the use of antiepileptic medications, such as carbamazepine and gabapentin, which must be customized and based on medical and psychiatric considerations (Johnson, 2019). Inappropriate treatment of epilepsy leads to several problems for patients, including low quality of life, higher risk of sudden death, and greater chances of developing physical, psychological, and social comorbidity conditions (Siarava et al., 2019;Tomson et al., 2008). When treated correctly, patients suffering from seizures manage to lead a normal life (Guerreiro, 2016).
Over the past decades, most of the development of anti-epileptic drugs has been based on the use of pre-clinical epilepsy models induced by pentylenetetrazole (PTZ) (Shimada & Yamagata, 2018), maximum electroshock (MES) (Socała et al., 2019) and on the use of pilocarpine (Kim & Cho, 2018). Although the number of antiepileptic medications available on the market has grown exponentially, the percentage of people with intractable seizures remains high (Z. Chen et al., 2018;Johnson, 2019) and issues associated with conventional treatments, specially drug resistance, are still frequent (more than 30% of individuals) (Chen et al., 2018;Mesraoua et al., 2019;Ryvlin, 2005). Although, several criteria have been adopted to classify drug resistance, a precise criterion has not been agreed on. A hierarchical level framework to characterize therapeutic interventions to trials has been suggested by Kwan et al (2010), where failure to at least two antiepileptic drug interventions, used alone or in combination, validates drug resistance (Kwan et al., 2010).
Lately, considerable attention has been given to the use of vegetal species as a new therapeutic approach for the treatment and control of recurrent seizures (Bulaj et al., 2016;Pina et al., 2020), including the use of plant extracts , essential oils (da Fonsêca et al., 2019) and isolated compounds, such as cannabidiol (Samanta, 2019).
Since ancient times, and yet aiming at establishing a balance between the physiological systems and providing healing, traditional Chinese medicine has used natural products to improve human health and well-being (Ma et al., 2016). Indeed, the application of these vegetal species has been remarkedly investigated and largely used to combat drug-resistant bacteria and viruses, infectious diseases, cancer, and as central nervous system disorders, including alzheimer, parkinson e recurrent seizures (Huyan et al., 2016;Makaremi et al., 2021;Marshall, 2020;Veeresham, 2012;Vilasboas-Campos et al., 2020;Vyas et al., 2019;. In contrast to conventional therapies, these products have lesser side effects and higher tolerability, making these products a promising therapeutic alternative in the search for new antiepileptic drug options (Deivasigamani et al., 2021;Popovych et al., 2020;Verma et al., 2021).
As these new natural therapeutic alternatives become more readily available (Pina et al., 2020), so does the need for further scientific discoveries and drug development as existing conventional drugs have only symptomatic effects. Pre-clinical animal model studies are essential for establishing efficacy and adjusting the safety profile of these novel substances prior their translation into clinical settings (Löscher, 2011(Löscher, , 2017. Thus, the objective of this review is to conduct a survey on the recent patent fillings of new therapeutic plant-based alternatives used for treating epilepsy and/or seizures, published between 2015 and 2020.

Methodology
The patent search for this review was carried out using one of the largest patents data base in the world (ESPACENET) in January 2021. The criteria search included patents published between 2015 and 2020 related to the use of anticonvulsant natural/pant-based products with clinically relevant activity in humans; and the International Patent Classification (IPC) A61P25/08 (antiepileptics, anticonvulsants) and A61K36 (medicinal preparations containing algae, lichens, fungi or plants or their derivatives) categories.
The following inclusion criteria were applied for the selection of patents: vegetal species (extracts, essential oil and compounds isolated from plants) with in vivo anticonvulsant effect studies applied to patients suffering from epilepsy and/or seizures. Patents in which mixtures of plants or extracts, combinations with other drugs and duplicates were excluded.

Results
Initially, 607 patents were found comprising the use of natural products and anticonvulsants. However, not all patents provided scientific evidence of an anticonvulsant effect. Thus, 16 patents were selected by read in full ( Figure 1). The selection showed that 2018 was responsible for the largest number of patent inventions filings, accounting for 31.25% (n = 5). Despite the year 2019 there was a decrease in the number of patent filings, 2020 appears with an increase, being the second year with the highest numbers of filings growth (n = 4; 25%), presenting a growth tendency as can be seen in Figure 2.  The middle-income country of China accounted for the majority of patent inventions related to innovative compounds based on vegetal species with anticonvulsant activity, accounting for 56.25% (n = 9) of the total published applications. The United Kingdom filed 31.25% (n = 5) of total patents found, followed by Spain and Russia with 6.25% (n = 1 each) ( Figure 3).
These findings could be related to the fact that approximately 80% of patients suffering from epilepsy are located in developing countries, including China (66%) and India (95%) (Ding et al., 2019), and hence resulting in an increased interest for finding effective new compounds. Determinants of pharmaceutical R&D research investments, which are based on proof of the effectiveness of traditional medicine, socioeconomic costs resulting from hospitalizations and consequences on the patient's quality of life, also would account to these results (Chatterjee et al., 2020;Ding et al., 2019;Yu et al., 2019). The university and business sectors were responsible for the majority of published applications with 37.5% (n = 6) and 31.25% (n = 5), respectively. These sectors mainly sought to investigate the effectiveness of using traditional medicine in humans and to propose new alternative products to standard drug-resistant epilepsy treatments (Auditeau et al., 2019;Mathon et al., 2020). A small number of patents were filed by individuals (25%, n = 4) and a minor share filed by university and companies' partnership (6.25%, n = 1).
All published patents in this study reasoned their invention findings and respective anticonvulsant effects based on pre-clinical studies. The most used models to screen for antiepileptic drugs were the convulsion induced by administration of PTZ (52.63%, n = 10), MES (15.79%, n = 3) and an association of pilocarpine and lithium chloride (15.79%, n = 3), which mimic generalized and focal seizures. The remaining models represent 15.79% (n = 1, each) and include febrile seizure, administration of kainate acid and strychnine.
This review reports 10 extracts, 1 essential oil and 8 isolated compounds from plants tested in seizure models (Table   1). Luo et al. (2015) obtained an extract from the dry root powder of the Salvia mitliorrhiza plant using ultrasound and acetone as the extraction solvent. Additionally, the authors have also identified the tanshinone IIA (6,6-Trimethyl-6,7,8,9tetrahydrofenantro [1,2-b] furan-10, 11-dione) using high-pressure liquid chromatography (HPLC). Tanshinone IIA is a multitarget drug known for its wide-ranging remedial effects, including protective properties in neurodegenerative diseases . Both the extract and the isolated compound were tested at doses of 0.1, 1.0 and 10 mg/kg, intravenous (i.v.), and demonstrated effectiveness against convulsions induced by PTZ infusion and corneal stimulation in mice. Due to their neuroprotective effects, these compounds can be used as an alternative antiepileptic drug treatment (Luo et al., 2015). In 2016, 3 patents using compounds initially obtained from ethanolic extracts were filed in China (Chen & Hu, 2016;Hu et al., 2016;Shan et al., 2016). Ethanol is a commonly used extraction solvent due to its safety and ability to extract a variety of active compounds from plants (Sabedra et al., 2017). Hu Kai and collaborators report the purification of aucubin, an iridoid glycoside obtained from the ethanolic extract of Eucommia ulmoides via ultrasound. Aucubin treatment treatment (5 and 10 mg/kg orally) administered for 14 days reduced neuronal death by reduced mixed lineage kinase domain-like (MLKL) domains and receptor-interacting protein kinase 1 (RIP-1) proteins. When induced by pilocarpine (20 mg/kg, intraperitoneal (i.p.)) and lithium chloride (125 mg/kg, i.p.), animals treated with aucubin increased the expression of beclin LC3B-1 and the levels of the structural proteins of autophagosomal membrane LC3B-II/I, suggesting a potential neuroprotective effect . Similarly, Yunnan Ruifen Biological Tech Co Ltd also discloses in their invention several methods for obtaining ethanol extract from Industrial hemp. Industrial hemp. is a plant of the species Cannabis sativa L. used in the industry due to its high nutritional value and potential functional characteristics (Farinon et al., 2020). Treatment administered at 20 mg/kg, i.p., of the ethanolic extract have demonstrated an increase in seizure latency and mortality latency in mice induced by PTZ (100 mg/kg, subcutaneous (s.c.)) (Chen & Hu, 2016).
Zhejiang Conba Pharmaceutical Co Ltd and the Zhejiang University of Technology report the obtention of Ginkgo biloba ethanolic extract with 11% lactones, 40% flavonoids and 48% procyanidins. Ginkgo biloba is a medicinal plant used in the treatment of Alzheimer's disease, cognitive impairment, diabetes, hypertension and dyslipidemia (Eisvand et al., 2020;G. Yang et al., 2016). The inventors were able to show that a more pronounced antiepileptic effect is achieved when the extract formulation has a higher content of procyanidin than the total flavonoids. Studies using this extract formulation for 4 or 8 weeks at 40 mg/kg, orally, also reduced the seizure incidence and increased the expression of proteins responsible for promoting differentiation of neuronal cells, including brain-derived neurotropic factor (BDNF), vascular endothelial factor (VEGF) and glial cell line-derived neurotrophic factor (GDNF), after administration of lithium chloride (3 mmol/kg, lumbar) and pilocarpine (20 mg/kg, lumbar) (Shan et al., 2016).
Following, in 2017 two patents employing ethanolic extracts and ultrafine powder have been disclosed (Guo et al., 2017;Ma et al., 2017). Guo Liangium and collaborators disclose the invention of a drug based on different methods of acquiring Monochasma savatieri Franch ethanol extract. Studies have shown that treatment with this ethanolic extract at doses of 1.25 to 3.25 g/kg, p.o. for 7 days reduced seizures and mortality in mice subjected to the administration of PTZ (100 mg/kg, s.c.) and MES (100 V for 0.3s) (Guo et al., 2017). Similarly, patent developed by Chengdu University of Traditional Chinese Medicine discloses a preparation of ultrafine powder from Gastrodia elata. Gastrodia elata is a traditional Chinese medicine known for its broad pharmacotherapeutic properties to treat central nervous system disorders, including epilepsy (Liu et al., 2018). Inventors claims include reduced seizure and mortality of animals when subjected to PTZ at 62.5 mg/kg, i.p. upon oral administration of the ultrafine powder at 0.3, 0.6, 1.2 g/kg doses, after a period of 5 days administration, (Ma et al., 2017).
Five patent applications were found in 2018 (Cai & Ran, 2018;Cao & Zhang, 2018;De Witte Petter et al., 2018;Stott et al., 2018;Whalley et al., 2018). Among the patents, the invention filed by De Witte Petter et al. ( 2018) reports the evaluation of the bisabolene bioactive compound isolated from turmeric oil through hydrodistillation in a Clevenger type apparatus. The authors state that the pharmacotherapeutic effect of bisabolene at doses of 2.5, 5 and 10 pg/mL appears to reduce induced convulsive movements after PTZ administration (20 mM) (De et al., 2018). Another patent invention disclosed, includes the obtention of the extract of Trifolium repens by decoction extraction method (Cao & Zhang, 2018). Trifolium repens is a plant with antioxidant, anti-inflammatory, analgesic and antimicrobial effects also used in traditional Chinese medicine (Ahmad & Zeb, 2020). The invention filed by the Zhejiang Chinese Medical University suggests administration of the extract for 3 days at doses of 20, 40 and 80 mg/kg, orally. The Trifolium repens extract formulation has proven to be effective against febrile seizures in rats (Cao & Zhang, 2018). Similarly, another patent application employing plant extracts in antiepileptic drugs includes the use of total flavonoids from the ethanolic extract of Tagetespatula L.. The use of these phytochemicals at 400 and 800 mg/kg, p.o. accounted for reducing the seizure and increasing the latency of death in mice also induced by PTZ administration (9 mg/mL, i.p.) (Cai & Ran, 2018).
Over the last few years increased interest in cannabis-based products has been observed within the clinical pharmacology sector for its properties in reducing the frequency of convulsive seizures (Perucca, 2017;Russo & Marcu, 2017). Two patents filed in 2018 by Gw Pharma Ltd refer to methods of obtaining purified cannabinoid compounds from Cannabis. Cannabidiolic acid (100 mg/kg) demonstrated anticonvulsant effects throughout increased seizure latency, as well as reducing severity and mortality in rats subjected to administration of PTZ (90 mg/kg, i.p.) (Stott et al., 2018). Similarly, the same company also report that the use of Cannabidivarin (100 and 200 mg/kg, i.p.), an analogue of cannabidiol, which also reduced seizure severity and mortality in the PTZ models (80 mg/kg, i.p.) .
In 2019, only one patent belonging to the Heilongjiang University of Chinese Medicine was found (B. Yang et al., 2019). The treatment with the Bupleurum chinense ethanol extract and its essential oil (obtained by steam distillation) at 200 and 400 mg/kg, i.p. reduced seizure and duration in experimental animal models after the administration of PTZ (60 mg/kg, ip) and kainate acid (0.5 μg/μL, i.c.v.). In addition, the extract and essential oil showed neuroprotective effects in hippocampal cells, suggesting that these compounds may be a promising alternative to the treatment of seizures (B. Yang et al., 2019).
Finally, in 2020, 4 patent inventions were found (Belousov et al., 2020;Geoffrey et al., 2020;Stott et al., 2020;. The first patent invention, deposited by the company Gw Res Ltd, claims the isolation for the first time of the compounds 7-hydroxy-cannabidol and 7-hydroxy-cannabidivarin (100 and 200 mg/kg, i.p.), which have shown to act on the nervous system increasing seizure latency time, and reducing the severity and mortality of animals submitted to PTZ (70 or 80 mg/kg, i.p.) (Stott et al., 2020). This same company, also obtained another cannabinoid compound derived from Cannabis, Cannabidiol-C4. Treatment of mice with Cannabidiol-C4 at 100 and 200 mg/kg, i.p. was responsible for reducing the number of seizures induced to electrical stimulation (Geoffrey et al., 2020).
Empetrum nigrum L. is a plant that has been widely employed in the treatment of epilepsy, paralysis and as a sedative in folk medicine (Jurikova et al., 2016). The third patent registered by Belousov Mikhail and colleagues, refers to a method to obtain an acetonic fraction from the dry chloroform extract of the plant Empetrum nigrum L. The preventive treatment with 150 mg/kg, orally, for 5 days, provided increased life expectancy and survival in mice induced with strychnine (1.5 mg/kg, s.c.) (Belousov et al., 2020). The last patent was filed by The Second Affiliated Hospital Of Xi'an Jiaotong University College Of Medicine and describes ways of obtaining the flavonoid fraction of the Stellera chamaejasme plant. The authors attest that the flavonoids present in S. chamaejasme are capable of reducing neuronal damage caused by the administration of Pilocarpine (30 mg/kg, i.p.) and lithium chloride (180 mg/kg. i.p.) in rats . Thus, the natural products obtained from plants are effective alternatives in the treatment of partial and generalized epilepsy.

Discussion
In this review, we assessed the published patent inventions referring to natural compounds in the form of extracts, essential oils and isolated compounds with proven anticonvulsant properties, filed between the years of 2015 and 2020. 10 patents (52.63%) reported methods of obtaining plant extracts, 1 patent (5.26%) disclosed an essential oil and 8 patents (42.11%) described purified plant compounds. These findings reinforce the idea that the use of natural products can be a promising alternative for the treatment of pathologies, including epilepsy (Elliott et al., 2020;Quintans-Júnior et al., 2008).
In view of the pharmacological potential of medicinal plants and the reduction of adverse effects, investment in the scientific production of these compounds has increased. Natural compounds derived from plants have been extensively studied and are commercially available. Sativex ® , for example, was the first medicine produced based on Cannabis. Despite being initially indicated for multiple sclerosis, studies have shown the effectiveness of its active principle on epilepsy (Filloux, 2015). Recently, another drug containing cannabidiol, produced in Brazil, with potential indication for the treatment of refractory epilepsy has been approved. Unlike Sativex ® , the formulation containing only cannabidiol has fewer side effects due to the absence of psychoactive substances, such as Δ 9 -THC (Gaston & Friedman, 2017).
Moreover, all purified cannabinoid compounds have been patented by the pharmaceutical industry Gw Pharma Ltd, which is a pioneer in the search for new pharmaceutical compositions derived from Cannabis. Phytocannabinoids cannabidiolic acid, cannabidivarin, 7-hydroxy-cannabidol, 7-hydroxy-cannabidivarin and cannabidiol-C4 act on cannabinoid receptor 1 (CB1) and cannabinoid receptor 2 (CB2) promotes reduction of neuronal excitability in the hippocampus. The biochemical interaction of these receptors results in the regulation and release of neurotransmitters, including glutamate present on the surface of brain cells (Gaston & Friedman, 2017;O'Connell et al., 2017;Rosenberg et al., 2017;Yao et al., 2019).
Although most of the patents in this review do not report the biochemical interactions of the compounds studied, a few mechanisms of action can be suggested. Salvia miltiorrhiza, for example, can exert its anticonvulsant effect through agonistic action on gamma-aminobutyric acid (GABAA) receptors (Lin et al., 2021). In addition, this specie can exert neuroprotective effects through the modulation of inflammatory mediators and oxidative stress (Su et al., 2015). Aucubin, studied in the patent by Hu Kai et al. (2016), is responsible for lessening neuronal damage by reducing Mixed Lineage Kinase domain-Like (MLKL) domains, RIP-1 receptors, Beclin-1 and LC3BII/LC3BI proteins and inflammation markers including tumor necrosis factor alfa (TNF-α) and interleukin 1 beta (IL-1β)) (S. Chen et al., 2019;Pina et al., 2020;J. Wang et al., 2017).
Similarly, Ginkgo biloba extract is also recognized for its neuroprotective ability to reduce neuronal damage and oxidative stress (Yan et al., 2020;D. Yu et al., 2020). Likewise, the anticonvulsant effect of Gastrodia elata extract studied in patent Ma et al. (2017) is known to alter the amount of GABA neurotransmitters receptors and to reduce neuronal damage induced in seizure models (Matias et al., 2016). The anticonvulsant effects of Monochasma sauatieri Franch extract claimed in patent Guo et al. (2017) can be related to its antioxidant ability to increase the enzymatic activities of superoxide dismutase (SOD) and glutathione (GSH), as well as to decrease the content of malondialdehyde (MDA) (Shi et al., 2013).

Conclusion
Thus, all of these compounds are promising alternatives due to their neuroprotective potential and mechanisms of action on GABAA, CB1 and CB2 receptors. In view of this, the development of new plant-based pharmaceutical compositions is a promising alternative for the treatment of epilepsy, especially for patients suffering from drug-resistant epilepsy. However, there are several limitations due to the initial state of these surveys. On the other hand, these findings are crucial for carrying out safety and efficacy studies using the tested substances.