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Gastrodia elata and epilepsy: rationale and therapeutic potential.

ABSTRACT

Background: Castrodia elata Blume (G. elata) is a traditional Chinese herb used for centuries in folk medicine. Due to the claimed anticonvulsant properties of C. elata, it is expected that this herb continues to be a target of research, aiming to deepen the available knowledge on its biological activity and safety. Purpose: The current review aims to discuss the most recent advances on the elucidation of the phytochemical composition and anticonvulsant potential of G. elata.

Methods: Available literature was reviewed from PubMed, ISI Web of Knowledge and Science Direct, using combinations of the following keywords: Castrodia elata, tianma, epilepsy, anticonvulsant and pharmacokinetics. Abstracts and full texts were evaluated for their clarity and scientific merit.

Results: G. elata rhizome, as well as specific phenolic compounds isolated from this herb, have demonstrated anticonvulsant potential in a variety of in vitro and in vivo models. The pharmacological mechanisms potentially involved in the anticonvulsant activity have been extensively studied, being similar to the known mechanisms claimed for the available antiepileptic drugs. In addition, the pharmacokinetics of the main bioactive component of G. elata (gastrodin) has also been studied.

Conclusion: Due to its recognised therapeutic properties, C. elata has gained an increasing interest within the scientific community and, therefore, new medicinal preparations containing G. elata rhizome itself or its bioactive components are expected to be developed in the coming years. Moreover, specific phytochemical constituents isolated from G. elata may also be considered to integrate programs of discovery and development of new anticonvulsant drug candidates.

Keywords:

Epilepsy

Gastwdia elata

Gastrodin

Phenolic compounds

Anticonvulsant properties

Introduction

Natural products have been widely used as medicines, dietary products and nutritional supplements since ancient times due to the fact that they are a rich source of bioactive compounds and multiple benefits for human health have also been shown. Effectively, the use of medicinal plants has been validated by traditional use and they are time tested when compared with modern medicinal herbal supplements (Atanasov et al., 2015). For this reason, herbal plants and some of their bioactive compounds have come to highlight their therapeutic potential and have been the subject of extensive research (Kim et al., 2014), mainly at the level of their biological activities and the underlying molecular mechanisms of action. Consequently, natural products can represent valuable starting materials for drug discovery programs (Bauer and Bronstrup, 2014).

Gastrodia elata Blume (G. elata) is a traditional herb that has been used in oriental countries for centuries (Kim et al., 2012a, 2003b). Its dry tuber is officially listed in the Chinese Pharmacopoeia and has been used as an anticonvulsant, analgesic and sedative product (Ojemann et al., 2006). However, it has been described as having a large variety of other pharmacological properties. Additionally, several phytochemical compounds isolated from this herb, such as gastrodin, 4-hydroxybenzyl alcohol (HBA), vanillin, vanillyl alcohol, 4-hydroxybenzaldehyde, [N.sup.6]-(4-hydroxybenzyl) adenine riboside (NHBA) and parishins, have been proposed as playing an important role in the pharmacological and therapeutic properties claimed for G. elata. The majority of studies in this context has been focused on the potential interest for central nervous system (CNS) disorders as it has been recently reported (Jang et al., 2015). For instance, the treatment of dopaminergic SH-SY5Y cells with an ethanol C. elata extract showed protective effect on l-methyl-4-phenylpyridinium-induced cytotoxicity (An et al., 2010) and similar results were observed with gastrodin Qiang et al., 2014) and vanillyl alcohol (Kim et al., 2011). In in vivo conditions, using a Parkinson's disease mouse model, an aqueous G. elata extract revealed better antidyskinetic effects than those observed with amantadine, a reference drug (Doo et al., 2014). Regarding Alzheimer's disease, the ethyl ether fraction of a methanol G. elata extract reduced the amyloid-[beta] peptide-induced cell death similarly to melatonin (Kim et al., 2003), and an aqueous extract improved cognitive functions in mouse and showed superior in vitro results than the observed with Gouteng herb and huperzine A (Mishra et al., 2011). Additionally, gastrodin decreased the area of amyloid-[beta] peptide deposition in the cortex and hippocampus of Tg2576 transgenic mice (Hu et al., 2014) and 4-hydroxybenzyl methyl ether exhibited memory-ameliorating effects on SCH23390- and scopolamine-induced memory impairment models (Lee et al., 2015). Considering the treatment of cerebral ischemia, it has been reported an important neuroprotective effect of vanillin and HBA against ischemic neuronal cell death in the hippocampal CAI region in gerbils subject to transient global ischemia (Kim et al., 2007). A long-term treatment of a rat model of Tourette's syndrome with gastrodin also revealed promising results, suggesting a dual restoring effect of gastrodin on the striatal dopamine content (Zhang and Li, 2015). Concerning psychiatric disorders, an aqueous extract of G. elata evidenced antidepressant-like effects possibly via regulation of serotonergic and dopaminergic systems (Chen et al., 2009) and this plant also showed potential benefits for the treatment of schizophrenia in the phencyclidine mouse model (Shin et al., 2010). An additional study also suggested anxiolytic-like effects for HBA and 4-hydroxybenzaldehyde (Jung et al., 2006) and NHBA appeared to potentiate the hypnotic effect of sodium pentobarbital in mice as an agonist for both adenosine [A.sub.1] and [A.sub.2A] receptors (Zhang et al., 2012).

Beyond the CNS effects of G. elata constituents, many other pharmacological and therapeutic properties have been under investigation. For example, an ethanol extract of G. elata (Ahn et al., 2007) and vanillyl alcohol Uung et al., 2008) inhibited the angiogenesis in the chick chorioallantoic membrane assay. Other G. elata extracts similarly showed potential in alleviating tumorigenesis and exhibited antimetastatic activity (Heo et al., 2007). In addition to its anti-angiogenic effects, vanillyl alcohol also inhibited the vascular permeability and exhibited anti-nociceptive activity probably through the involvement of prostaglandin biosynthesis (Jung et al.. 2008). Moreover, vanillin was found to be a potent anti-inflammatory and analgesic compound, and 4-hydroxybenzaldehyde, HBA and benzyl alcohol significantly inhibited the cyclooxygenase-1 and cydooxygenase-2 activities (Lee et al., 2006). The cardioprotective effects of G. elata have also been studied, mainly focusing on blood pressure and serum lipid levels, which were reduced by acidic polysaccharides purified from Castrodia rhizomes in spontaneously hypertensive rats (Lee et al., 2012) and Sprague Dawley rats (Kim et al., 2012b). A reduction in insulin resistance was also observed in diet-induced obese rats treated with an aqueous G. elata extract, and this effect was mainly attributed to the phenolic compounds 4-hydroxybenzaldehyde and vanillin (Park et al., 2011). Furthermore, gastrodin significantly prolonged the coagulation time and decreased the fibrinogen content, suggesting its possible use as a promising anticoagulant lead compound (Liu et al., 2006); gastrodin also inhibited the cardiac hypertrophy induced by pressure overload in mice (Shu et al., 2012). A methanol extract of G. elata also presented gastroprotective effects (An et al., 2007) and, more recently, the anti-osteoporosis activity of gastrodin has also been explored (Chen et al., 2015a; Huang et al., 2015a).

The clinical evidence about the therapeutic properties of G. elata and/or its phytochemical constituents is still scarce. However, a double-blind, placebo-controlled clinical trial was already performed, suggesting an anti-sickling effect of vanillin (Garcia et al., 2005). Another double-blind, randomized, controlled clinical study was carried out to investigate the preventive effects of gastrodin on the neurocognitive decline, a common complication after cardiac surgery with cardiopulmonary bypass; this study showed a significant decrease in the neurocognitive decline in the patients treated with gastrodin (Zhang et al., 2011). Moreover, a clinical study is ongoing to evaluate whether G. elata is effective in the treatment of masked hypertension (ClinicalTrials.gov, 2016).

Among the multi-pharmacological effects of G. elata and its bioactive constituents on the CNS, the anticonvulsant effects are certainly worthy of note (Ojemann et al., 2006). Indeed, epilepsy is one of the most common serious neurological disorders, affecting around 60 million people worldwide (Shetty and Upadhya, 2016); moreover, despite the large arsenal of antiepileptic drugs (AEDs) currently available, approximately 30-40% of patients develop pharmacoresistance (Kwan et al., 2010; Sorensen and Kokaia, 2013), thus existing an imperative need of new AEDs with improved efficacy. Furthermore, over the years, patients with epilepsy have used a variety of herbs to treat known comorbidities of epilepsy and common adverse events of AEDs (Ekstein, 2015), and some medicinal plants have shown potential as new treatment options for patients whose seizures are uncontrolled with the available AEDs. Actually, extracts of plants and/or their single constituents have shown to act on the same pharmacological targets as those of the most commonly used AEDs (Sucher and Carles, 2015; Zhu et al., 2014). Undoubtedly, the search for new anticonvulsant lead compounds among phytochemicals constituents has emerged as a promissory and alternative drug discovery approach. For instance, according to the progress report on new AEDs published as a summary of the Twelfth Eilat Conference (EILAT XII) that took place in Madrid (Spain) in 2014 (Bialer et al., 2015), at least three of the AED candidates in clinical development are herb-derived compounds (cannabidiol, cannabidivarin and huperzine A). Therefore, taking into account the contribution of Ojemann and collaborators (Ojemann et al., 2006) and the new literature available on the anticonvulsant properties of G. elata extracts and some of their phytochemical constituents, this review intends to address in a more extensively manner the phytochemical composition of G. elata, as well as discuss the more recent data on the pharmacokinetics and the scientific evidence for potential therapeutic benefits in epilepsy.

Methods

To prepare this review, an extensive literature search from three databases, PubMed, ISI Web of Knowledge and Science Direct, was performed to generate a critical but comprehensive overview of the phytochemistry, pharmacokinetics, pharmacological and anticonvulsant properties exhibited by crude extracts or purified compounds of G. elata. The keywords for the search consisted of combinations of the following terms: Castrodia elata, tianma, epilepsy, anticonvulsant and pharmacokinetics.

Botanical aspects

G. elata, commonly known as "Tianma" (Ojemann et al., 2006), is a member of the Orchidaceae family (Tao et al., 2011). Although the orchids usually are famous due to their beautiful flowers, Gastrodia species have attracted attention because of their pharmacological properties. Gastrodia plants, lacking green leaves and chlorophyll, are saprophytic perennial herbs comprising approximately twenty species worldwide. These species grow in the glades or at the edge of forests in humid mountain areas at an altitude of about 400-3200 m, and the populations are isolated by lowlands with different environmental conditions (Chen et al., 2014a). Specifically, G. elata is found primarily in eastern Asia and in the mountainous ranges of China and Korea (Ojemann et al., 2006). This species exhibits high level of genetic diversity, which was mainly attributed to its perennial habit and mixed reproduction system (Chen et al., 2011a). As this herb is entirely dependent upon the fungus Armillaria mellea for nutriment (Muszynska et al., 2011), the cultivation of this plant outside its native region remains a challenge. In addition, because of its over-collection as food and for medical nutrition therapy, it is becoming increasingly rare in the wild state and, therefore, G. elata has been currently listed as a rare and endangered plant species in China and even in the world (Chen et al., 2011a). Thus, great efforts towards its preservation based on genetic studies (Chen et al., 2014a, 2014b), as well as in new methods of cultivation have been under investigation (Huang et al., 2011).

Phytochemistry

Over the years, several phytochemical studies have been mainly focused on the isolation and identification of the phenolic elements of G. elata. Indeed, phenolic compounds constitute the majority of the total constituents of G. elata and they are usually considered responsible for the main pharmacological and therapeutic properties ascribed to this medicinal plant. This can be explained in part by the fact that phenolic compounds are well recognized for their antioxidant activity (Kancheva and Kasaikina, 2013), combating the reactive oxygen species which, when produced in excess, are associated with several physiological and pathological conditions (Rosenfeldt et al., 2013). Therefore, several studies aiming at evaluating the antioxidant activity of the compounds present in G. elata have been carried out, focusing just on the antioxidant properties and/or assessing the impact of this antioxidant activity in other health conditions in which oxidative stress is suspected to play an important role, such as neurodegenerative disorders (Hwang et al., 2009; Jung et al., 2007; Yu et al., 2005). Overall, such compounds include simple phenols and phenolic conjugates such as parishins. In summary, the compounds isolated from G. elata are listed in Table 1.

Gastrodin--the main bioactive compound of G. elata

Gastrodin (p-hydroxymethylphenyl-[beta]-D-glucopyranoside) (Fig. 1) is a simple phenolic glycoside and it was the first bioactive compound isolated from C. elata (Ojemann et al., 2006). It is considered the main and most important bioactive component extracted from this herb (Li et al., 2001), having multiple beneficial properties. The importance of gastrodin in the phytochemical composition of G. elata is highlighted by the fact that the content of gastrodin is assayed as one of the most important phytochemical markers in the quality standardization of G. elata tubers (Tao et al., 2009).

Gastrodin is usually obtained by extraction from G. elata or by chemical synthesis. Both of these methods are not straightforward, being expensive and leading to many by-products and serious pollution problems as well as wasting of natural resources. Thereby, new methods for the synthesis of gastrodin have been developed over the years. In this context. Zhu and collaborators reported the purification of the enzyme responsible for the microbial transformation of 4-hydroxybenzaldehyde into gastrodin from the fungal strain Rhizopus chinensis SAITO AS3.1165 (Zhu et al., 2010). In addition, the biotransformation of exogenous HBA to gastrodin using hairy root cultures of Datura tatula L inoculated with Agrobacterium rhizogenes is also a promising approach, with the efficiency of HBA glycosylation reaching approximately 60% (Peng et al., 2008). Using the same precursor, it was also demonstrated the production of gastrodin by the fungi Penicillium cyclopium AS 3.4513 with a yield of 65 mg/l (Fan et al., 2013).

General phenolic compounds

Although gastrodin has been considered the major active component responsible for the medicinal properties of G. elata, diverse reports have suggested that the pharmacological effects of this herb cannot only be attributed to this compound alone (Yang et al., 2007). In fact, other bioactive compounds can be isolated from this herb (Hayashi et al., 2002) and can also be responsible for several bioactivities. In this context, a highlight goes to HBA, a gastrodin metabolite, which is also named gastrodigenin (Ojemann et al., 2006). The structures of these compounds and of other main simple phenolic constituents that have been isolated and whose pharmacological and therapeutic activities have been studied are exhibited in Fig. 1.

Phenolic compounds containing a nucleoside

The NHBA (Fig. 2) is a prominent compound isolated from G. elata, which has anxiolytic and sedative properties, among others. In fact, several efforts have been developed towards the effective semisynthesis of this compound (Huang et al., 2007) and to the study of its in vivo metabolic pathways, identifying the major metabolites in rat urine and plasma after oral administration (Lei et al., 2011). Additionally, several NHBA derivatives were synthesized and evaluated for Huntington's disease (Chen et al.. 2011b) and the 3-methoxy-NHBA [[N.sup.6]-(3-methoxy-4hydroxybenzyl) adenine riboside] was also tested for the treatment of insomnia (Shi et al., 2014).

Phenolic conjugates containing a citrate moiety

A group of phenolic conjugates containing a citrate moiety (Fig. 3) has also been discovered in the composition of C. elata such as the parishins and a citryl glycoside, trimethylcitryl-[beta]-D-galactopyranoside, which is constituted by a sugar unit identified as galactose and an aglycone moiety characterized as a citric acid derivative (Choi and Lee, 2006). Interestingly, parishins can function as a gastrodin source through their metabolization after gastric absorption and, for this reason, they also have to be considered in the studies that investigate the pharmacological activities of this plant.

Polysaccharides

Other compounds isolated from the C. elata rhizome belong to the group of polysaccharides and it was found that the presence of these molecules can be related to the fact that the nutrition needed for C. elata growth is mostly dependent on the fungus Armillaria mellea (Muszynska et al., 2011). The polysaccharides isolated from this plant consist of only glucose molecules and their structures are represented in Fig. 4. Although we have not found reports of any polysaccharides isolated from G. elata associated with anticonvulsant properties, they have other biological activities, being mainly studied in the prevention of cardiovascular risk (Lee et al., 2012; Ming et al., 2012). Also, structureactivity relationship studies were carried out regarding their anti-dengue virus bioactivity (Qiu et al., 2007) and anti-angiogenic effects (Chen et al., 2012).

Pharmacokinetics

Up to date, a limited number of studies has been performed in order to assess the absorption and biodisposition of G. elata constituents. Even so, there are some non-clinical and clinical studies that reported the pharmacokinetics of gastrodin and its metabolite HBA in in vivo conditions. For instance, after intravenous (i.V.) gastrodin administration (50mg/kg) to Sprague-Dawley rats, it was observed that the parent compound (gastrodin) was rapidly distributed to the bile and brain, and quickly biotransformed to HBA. In fact, HBA was found in the bile and brain at 10 min post-dose and its levels rapidly declined after gastrodin i.v. injection. The gastrodin in blood reached a peak concentration of 24.1 [micro]g/ml whereas HBA attained a peak concentration of approximately 220-fold lower (0.109 [micro]g/ml) at 15 min post-dose. The results of this pharmacokinetic study also suggested that the brain exposure to gastrodin far exceeds the brain exposure to HBA (more than 8.7-fold at 15 min after administration). Nevertheless, taking into consideration the relationship between the systemic concentration levels of both compounds, it can be inferred that the metabolite HBA is able to pass through the blood-brain barrier in a more efficient manner than gastrodin. Additionally, using microdialysis probes, it was demonstrated that the recovery of both compounds (gastrodin and HBA) in bile is higher than 90%; these findings suggested the hepatobiliary system as the major route of gastrodin and HBA excretion (Lin et al., 2008). According to the study of Lin et al. (2007), the gastrodin brain-to-blood distribution ratio (k value) was also found to be very low, being 0.007 [+ or -] 0.002 and 0.01 [+ or -] 0.002 at doses of 100mg/kg (i.v.) and 300mg/kg (i.v.), respectively. Moreover, at this studied dose range (100 and 300mg/kg, i.V.), the distribution and elimination processes of gastrodin in rat seem to follow a linear kinetics (Lin et al., 2007). On the other hand, Wang et al. (2008) also studied the gastrodin biodisposition in the rat after the administration of 200 mg/kg (i.V.); the results obtained have also shown that the entry of gastrodin into the brain is rapid, but the extent of brain exposure was relatively small in comparison with the extent of total systemic exposure, as assessed by the area under the concentration-time curve (AUC). Indeed, the [AUC.sub.brain]/[AUC.sub.plasma] ratios were not high; more specifically, the individual ratios of the AUC in the cerebrospinal fluid, frontal cortex, hippocampus, thalamus and cerebellum to the AUC in the plasma were 4.8 [+ or -] 2.4%, 3.3 [+ or -] 1.2%, 3.0 [+ or -] 0.7%, 3.3 [+ or -] 1.3% and 6.1 [+ or -] 1.9%, respectively. As demonstrated from these neuropharmacokinetic data, the cerebellum was the brain region with a higher exposure to gastrodin, which may suggest that this compound may have more potent effects on cerebellar targets than in other brain areas. In this study, it was also shown that HBA is immediately formed after i.v. gastrodin administration, but the measured concentrations were very low and declined very quickly. Specifically, the HBA concentrations were inferior to the lower limit of quantification (LLOQ) of the bioanalytical method in plasma (LLOQ=0.15 [micro]g/ml) and in cerebrospinal fluid (LLOQ=0.07 [micro]g/ml) at 60 min and 90 min after dosing, respectively. Bearing in mind the pharmacokinetic data provided by all these studies (Lin et al., 2008, 2007; Wang et al., 2008), it should be highlighted that gastrodin undergoes a rapid biodistribution in rat, reaching quickly the CNS but the concentration levels achieved therein are much lower than those attained in blood. These findings suggest that the relatively small amount of gastrodin that reaches the brain may be enough to elicit the significant pharmacological effects in CNS ascribed to C. elata. Nevertheless, more studies are necessary to better elucidate what is the bioactive component that reaches the brain, as well as the contribution of the main gastrodin metabolite (HBA) for the claimed therapeutic properties. Additionally, after oral (p.o.) administration of different preparations of C. elata to rats, the absorption of gastrodin was also shown to be fast, with peak concentrations in plasma occurring at 10-20 min post-dose (Zheng et al., 2011).

The compound parishin, extracted from the roots of G. elata, was also studied and, actually, this component can be considered a gastrodin prodrug. In fact, after i.v. injection of parishin (116mg/kg) to Sprague Dawley rats, the compound was converted in approximately 50% to gastrodin, its main metabolite. The elimination half-life ([t.sub.1/2]) of parishin was low (0.29 [+ or -] 0.11 h), which suggests a fast degradation and, interestingly, the gastrodin [t.sub.1/2] (1.17 [+ or -] 0.34 h) after the administration of parishin was similar to that observed after the injection of gastrodin at 64.5mg/kg (1.31 [+ or -] 0.05 h) (Tang et al., 2015b). The same authors also intended to compare the pharmacokinetic parameters of gastrodin, parishin and the ethyl acetate fraction of ethanol extract of G. elata, considering that the last two (parishin and G. elata extract) are gastrodin sources in vivo. Interestingly, they discovered that these parameters are very different depending on the administration route studied. In this study it was found that the AUC (57.92 [+ or -] 11.94 [micro]g h/ml) of free gastrodin was higher than the AUC of gastrodin originated from parishin (4.96 [+ or -] 0.53 [micro]g h/ml) and from the G. elata extract (36.38 [+ or -] 3.84 [micro]g h/ml) and the peak plasma concentration ([C.sub.max]) was higher and had been reached earlier for free gastrodin ([C.sub.max] = 44.84 [+ or -] 14.51 [micro]g/ml at 0.42 h versus [C.sub.max]=3.47 [+ or -] 1.58 [micro]g/ml at 0.83 h from parishin and [C.sub.max] = 14.18 [+ or -] 3.94 [micro]g/ml at 1 h from the G. elata extract) after intragastric administration. However, through the same route the free gastrodin was faster eliminated, which was visible in its [t.sub.1/2] (1.13 [+ or -] 0.06 h versus 3.09 [+ or -] 0.05 h from parishin and 7.52 [+ or -] 1.28 h from the G. elata extract). A possible explanation for this is the fact that parishins B and C, which already are metabolites of parishin, can be further metabolized to gastrodin, prolonging the in vivo gastrodin levels (Tang et al., 2015a). Indeed, these results are very interesting, alerting to the point that the pharmacological activities claimed for parishins in in vivo conditions could actually be mediated by gastrodin or, ultimately, by HBA.

As it is believed that the main therapeutic actions of G. elata are exerted in the CNS, the intranasal route can be a promising alternative to traditional routes of administration due to the possibility to circumvent the blood-brain barrier in the drug delivery to the brain. In fact, intranasal gastrodin administration (50 mg/kg) provided an AUC in the cerebrospinal fluid comparable to that obtained by i.v. administration. Additionally, cerebrospinal fluid concentrations of gastrodin 60 min following intranasal administration were always higher than those achieved after i.v. administration, which suggested a direct nose-to-brain pathway to transport gastrodin from the nasal cavity to the brain (Wang et al., 2007). Later, a safe and stable in situ gel preparation was formulated for the effective nasal delivery of gastrodin in rats (Cai et al., 2011).

Undoubtedly, the data available on the pharmacokinetics of gastrodin at the clinical level are still quite scarcer than those generated from non-clinical conditions. Up to date, to the best of our knowledge, the pharmacokinetic analysis of gastrodin was only considered in one clinical study, which included eighteen male subjects to whom a gastrodin capsule was orally administered at a dosage of 200 mg. According to the obtained results, gastrodin was rapidly absorbed into blood ([t.sub.1/2Ka] = 0.18 h) and reached a peak concentration at 0.81 h. In humans, the gastrodin concentrations achieved in plasma also declined very rapidly, probably as a result of the fast distribution and elimination processes (Ju et al., 2010), similarly to what was previously referred in the rat.

Anticonvulsant properties and putative underlying mechanisms

As aforementioned, G. elata has been traditionally used to treat epilepsy in oriental countries and its anticonvulsant properties have been widely studied. Thus, in this section, the available scientific evidence on the anticonvulsant activity of G. elata extracts and its bioactive components, as well as their putative mechanisms of action, is critically discussed. In addition, a summary of the main in vivo and in vitro studies that support this activity is presented in Tables 2 and 3, respectively.

G. elata rhizome extracts

The rhizomes of G. elata have been used to prepare different aqueous and organic (e.g. methanol, ethanol) extracts (Yang et al., 2007). Therefore, it is not surprising the reference to various G. elata extracts (aqueous, methanol, ethanol), or even to fractions of such extracts, in the multiple biological evaluation studies already carried out (Heo et al., 2007; Park et al., 2011).

In general, the methanol extract of G. elata itself or different fractions of this extract (n-butanol and ether fractions) appear to be the most strongly associated with the anticonvulsant activity ascribed to this herb. This can be related with the presence and amount of some bioactive components in this type of extract, which may clearly depend on the way the final G. elata herbal preparations are obtained (Yang et al., 2007). In this context, Shin et al. (2011) observed that a methanol extract of G. elata significantly delayed the seizure onset time and shortened the seizure duration in a mouse model of acute cocaine-induced seizures (Table 2, entry 1). A possible mechanism of action that could explain such anticonvulsant activity was also explored in the same study. Indeed, experiments showed that the anticonvulsant activity mediated by the G. elata methanol extract was significantly reversed by the [GABA.sub.A] receptor antagonist bicuculline (0.25 or 0.5 mg/kg, i.p.) in a dose-related manner, but not by the [GABA.sub.B] receptor antagonist SCH 50,911 (1.5 or 3.0 mg/kg, i.p.). These results suggest that GABA receptors, particularly the [GABA.sub.A] receptor subtype, are implicated in the G. elata-mediated anticonvulsant protection against cocaine-induced seizures (Fig. 5). The same research group also evaluated this G. elata extract in a chronic mouse model of epilepsy induced by the administration of cocaine (15 mg/kg/day, i.p.) once every 2 days during 12 days, followed by 7 days of withdrawal before the behavioural sensitization test; then, the methanol extract of G. elata was given at 500 and 1000 mg/kg/day, p.o., during 20 days. Following this experimental protocol, although the G. elata extract in both doses did not attenuate the behavioural sensitization, it was able to block the conditioned place preference induced by cocaine (Shin et al., 2011). Additionally, a different mechanism was explored, being demonstrated that the methanol extract of G. elata appeared to have neuroprotective effects by reducing the toxicity induced by glutamate in HT22 cells (Table 3, entry 1). These outcomes were proposed to be related with the up-regulation of the phosphatidylinositol-3-kinase signalling pathway and the antioxidant activity exhibited by this extract in the reduction of the levels of reactive oxygen species induced by glutamate (Han et al., 2014).

Regarding the anticonvulsant protection manifested by different fractions of methanol extracts, it was found that the treatment with repeated doses of an ether fraction of the methanol extract of G. elata, during 14 days showed neuroprotective and anticonvulsant activity in the kainic acid (KA)-treated mouse model (Fig. 5). Indeed, the ether fraction of methanol extract of G. elata at the dose of 500 mg/kg delayed the onset time of neurobehavioural changes and reduced the severity of KA-induced seizures, as well as the hippocampal neuronal damage in the CAI and CA3 regions (Table 2, entry 2) (Kim et al., 2001). The same fraction was also evaluated in a subcutaneous pentylenetetrazole (scPTZ)-seizure model. In this assay, scPTZ was administered daily to rats, during 3 days, at a convulsive dose of 70 mg/kg. The recovery time was significantly reduced by the extract of G. elata as well as the severity of the seizures, which was calculated by the formula [SIGMA] [(degree of convulsion) x (frequency of convulsion for each degree)]/total frequency of convulsion (Table 2, entry 3; Fig. 5). Then, in a chronic model of epilepsy consisting in the administration of a subconvulsive dose (25 mg/kg) of scPTZ injected during 8 weeks, the pre-treatment with the ether fraction of the methanol G. elata extract lead to an increase on the GABA content levels in the brain of scPTZ-treated rats (Table 2, entry 4) (Ha et al., 2000). In another research work, among different G. elata studied extracts, the n-butanol fraction of the methanol extract seems to be the most effective in the inhibition of GABA-transaminase (GABA-T; Fig. 5), one enzyme involved in GABA degradation (46.48 [+ or -] 5.24%) (Choi and Lee, 2006).

Considering the KA-induced seizures in Sprague-Dawley rats, the pre-treatment with an aqueous extract of G. elata also appeared to reduce the number of three types of seizure attacks including wet dog shakes, paw tremor, and facial myoclonus (Table 2, entry 5). In the same study, it was demonstrated that the pretreatment with G. elata activated the c-Jun N-terminal kinases signalling pathway and c-Jun expression. On the other hand, the post-treatment with this herbal extract suppressed both the c-Jun N-terminal kinases signalling pathway and the c-Jun expression induced by KA (Table 2, entry 6). These results led the authors to suggest that the modulation of activator protein 1 expression via c-Jun N-terminal kinases signalling pathway by C. elata can contribute to the anticonvulsant effect of this herb (Hsieh et al., 2007). In order to understand the putative underlying mechanisms of action of C. elata, Hsieh and collaborators also reported that an ethanol extract of G. elata tested against neuronal damage in KA-treated Sprague-Dawley rats indicated a reduction of microglia ac tivation and a suppression of the neuronal nitric oxide synthase (Table 2, entry 7) (Hsieh et al., 2005).

Gastrodin and HBA

Although the G. elata extracts have been frequently evaluated in a range of different in vivo and in vitro assays, it is important to remember that these herbal preparations consist in very complex mixtures of bioactive components. Thus, with this kind of experiments, it is very difficult to understand which bioconstituents are responsible for the claimed pharmacological activities for the extracts. Therefore, as it is known the phytochemical profile of the herbal extracts, it is common to advance with the study of the properties of isolated biocompounds (e.g. gastrodin). Due to the fact that gastrodin is considered the main bioactive C. elata component, several studies have focused on the evaluation of its biological activities either after purchase or directly isolated from G. elata. Regarding its anticonvulsant properties, gastrodin, isolated from the n-butanol fraction of a methanol extract of C. elata, appeared to irreversibly inactivate the succinic semialdehyde dehydrogenase (SSADH) of bovine brain in in vitro conditions (Table 3, entry 2; Fig. 5) (Baek et al., 1999), thus preventing the GABA degradation in the synaptic cleft. Later, a more complete in vivo study evidenced that gastrodin isolated from the same extract was able to decrease the immunoreactivities of the three enzymes involved in GABA degradation (GABA-T, SSADH and succinic semialdehyde reductase (SSAR)] (Table 2, entry 8; Fig. 5). The findings obtained in this work also showed that the GABA-synthetic enzymes (two isoforms of glutamic acid decarboxylase--GAD65 and GAD67) and GABA transporters are not involved in the potential anticonvulsant mechanisms of action of gastrodin. Additionally, gastrodin also reduced the seizure score in a genetic seizure model (seizure-sensitive gerbils stimulated by vigorous stroking of the back with a pencil) (Table 2, entry 8) (An et al., 2003).

As it is widely recognised, monotherapy is considered the ideal pharmacological approach in epilepsy treatment because of reduced side effects, absence of drug interactions and better patient compliance. However, due to the availability of multiple AEDs with different mechanisms of action the possibility of "rational polytherapy", taking advantage of possible synergism, is often an option in patients with resistant epilepsy (Santulli et al., 2016). Following this idea, a recent study was designed to evaluate the anticonvulsant and neuroprotective effects resulting from the co-administration of gastrodin and Phenytoin against seizures induced by penicillin in mice; in the acute anticonvulsant experiments, both the gastrodin alone ([ED.sub.50] = 950.60 mg/kg) and the Phenytoin alone ([ED.sub.50] =45.50 mg/kg) showed anticonvulsant activity, but it was found that gastrodin and Phenytoin combination therapy can enhance the anticonvulsant effect and reduce the side effects of Phenytoin; the ideal phenytoin:gastrodin ratio was found to be 1:50 with an [ED.sub.50] value of 8.59:429.27 mg/kg. This fact means that the dose of Phenytoin was reduced by 81% and gastrodin by 55% without affecting the anticonvulsant activity (Table 2, entry 9). Additional chronic anticonvulsant experiments allowed assessing therapeutic and neuroprotective actions, as well as side effects of the phenytoin/gastrodin co-administration. As it is presented in Table 2 (entry 10), the combination therapy protected the normal balance and memory function of the mice that were compromised by Phenytoin and exhibited neuroprotective effects in the hippocampus (the neuron morphology was preserved and the number of surviving neurons was higher than the control group) (Zhou et al., 2015).

Although there are many studies reporting a wide spectrum of biological activities of the gastrodin metabolite (HBA), in a preliminary GABA-T inhibition assay it was found that this bioconstituent isolated from the ether fraction of the methanol extract of G. elata exhibited a very weak GABA-T inhibitory activity at a concentration of 10 [micro]g/ml (Ha et al., 2001). Additionally, Choi and Lee (2006) also evaluated HBA in the same assay (Table 3, entry try 3) (Choi and Lee, 2006) and, interestingly, the observed results confirm its weak inhibitory activity against GABA-T previously reported by Ha et al. (2001).

4-Hydroxybenzaldehyde and analogues

A study carried out by Ha and collaborators (Ha et al., 2001) revealed that 4-hydroxybenzaldehyde, isolated from the ether fraction of a methanol extract of G. elata, potently inhibited the activity of GABA-T in in vitro conditions; a similar effect was exhibited by 4-hydroxybenzaldehyde analogues containing an aldehyde group attached to the phenyl ring. The observed [IC.sub.50] values are present in Table 3 (entries 4-7), and it was clear that the inhibitory potency found for these compounds on the activity of GABA-T (Fig. 5) is significantly higher than that obtained for vigabatrin, a non-classic AED whose mechanism of action is thought to involve the potentiation of GABAergic neurotransmission by acting as an irreversible inhibitor of GABA-T (Krasowski, 2010). Ha et al. (2001) also assessed the effect of these compounds in the benzodiazepine receptors using a selective antagonist ([[sup.3]H]Ro15-1788) and a selective agonist ([sup.3]H]flunitrazepam). The results showed that the compounds under evaluation (Table 3, entries 4-7) did not have agonistic activity on the [GABA.sub.A] receptor complex (Ha et al., 2001). Indeed, 4-hydroxybenzaldehyde (5 x [10.sup.-6]g/ml) had already shown a GABA-T inhibitory activity higher than valproic acid (5 x [10.sup.-5]g/ml) (Ha et al., 2000). Furthermore, structure-activity relationship analysis on 4-hydroxybenzaldehyde derivatives as GABA-T and SSADH inhibitors indicated that a carbonyl or an amino group as well as a hydroxyl group at the para-position of the benzene rings are important for the inhibition of both enzymes. However, the introduction of an alkyl group at the same position may reduce their potency. Hence, the inhibition of these enzymes by the competitive inhibitors 4-hydroxybenzylamine and 4-hydroxybenzaldehyde (Table 3, entries 8 and 9) could result from the structural similarity between both molecules and the two endogenous enzymes' substrates (GABA and succinic acid), together with the conjugative effect of the benzene ring (Fig. 5) (Tao et al., 2006).

Vanillin and vanillyl alcohol

In the evaluation of the anticonvulsant effects of vanillyl alcohol on ferric chloride-induced seizures, it was observed that the pre-treatment with this G. elata biocomponent was able to reduce the counts of wet dog shakes, contrarily to the AED Phenytoin. Moreover, the authors suggested that this effect could be related with the antioxidant activity of vanillyl alcohol (200 mg/kg) in the rat brain, which was stronger than the verified with Phenytoin, in both right and left brain hemispheres (Table 2, entry 11) (Hsieh et al., 2000). Additionally, vanillin can allosterically modulate the GABAergic neurotransmission by enhancing the binding of the endogenous receptor agonist (Ha et al., 2001). The glutamate-induced cellular apoptosis and the increase in intracellular calcium induced by glutamate were also assessed in 1MR-32 human neuronal cells as possible anticonvulsant mechanisms of action of vanillin and 4-hydroxybenzaldehyde (Table 3, entries 10 and 11). Effectively, these G. elata constituents significantly inhibited both the intracellular calcium rise and the apoptosis induced by the excitatory neurotransmitter glutamate. In this study, similar results were achieved when the cells were treated with EGTA (1 mM), an extracellular calcium chelator (Lee et al., 1999); however, in this study, it would also have been interesting to test as a possible positive control a known AED acting as a calcium channel blocker and/or interfering with glutamatergic system pathways. The vanillin anticonvulsant activity was also evaluated through a model involving the electrically induction of seizures (fully amygdala-kindled seizures), in which the chronic state of epilepsy phenotype is established by repeated application of sub-convulsive electric stimulation. In this model, the pre-treatment with vanillin (administered intraperitoneally to rats 1 h before stimulation), suppressed the stage 5 seizures with the median effective dose ([ED.sub.50]) of 286 mg/kg; a similar result was achieved with Phenytoin at 50 mg/kg (i.p.). Moreover, vanillin also induced a significant shortened epileptic afterdischarge duration (Wu et al., 1989).

Other bioactive constituents

The biocomponent trimethylcitryl-[beta]-D-galactopyranoside, isolated from the n-butanol fraction of the methanol extract of G. elata, was evaluated in the in vitro GABA-T assay. Although some degree of GABA-T inhibition was evidenced by this compound (Table 3, entries 12; Fig. 5), it was not as effective as valproic acid (Choi and Lee, 2006).

In another study, the compound S-(4-hydroxybenzyl)glutathione, isolated from an aqueous extract of G. elata, was tested in the competitive binding assay with radiolabeled KA to the glutamate receptor (Fig. 5), and it had a slightly lower affinity than the observed with glutamate and glutathione (Table 3, entry 13) in the cortex of male Wistar rats (Andersson et al., 1995).

Critical opinion

Nowadays, the majority of molecules tested as new AED candidates follows the Anticonvulsant Screening Program of the National Institute of Neurological Disorders and Stroke of the U.S. National Institutes of Health. In fact, after its establishment in 1975, this program has made relevant contributions regarding the development of new AEDs, such as topiramate, lacosamide and retigabine, which were approved for epilepsy (NINDS, 2016). Although this program is frequently used for the identification of new chemical entities, it was surprising the scarcity of information found for the extracts and/or biocomponents isolated from G. elata in the first line of animal models usually used to predict the anticonvulsant activity (maximal electroshock seizure test and scPTZ test). In fact, there is only a research work that reports the anticonvulsant activity of the ether fraction of methanol extract of G. elata using an acute and chronic animal model of scPTZ (Ha et al., 2000); however, the use of an electrical animal model of seizures (e.g. maximal electroshock model or 6 Hz) was not found in the literature.

Even though several studies have focused on the mechanisms of action (the so-called "advanced studies") of the extracts and/or bioconstituents of G. elata, usually in research programs of discovery of new anticonvulsant compounds these mechanistic assays are considered at more advanced stages in order to explain the efficacy and/or toxicity data previously obtained in animal models of seizures (acute models) and epilepsy (chronic models). This is due to the fact that the in vitro studies themselves do not give enough information about the efficacy of the compounds as anticonvulsant drug candidates. Therefore, further investigation is required to explore the effective anticonvulsant potency of several of the discussed phytochemicals contained in the G. elata. For instance, taking into account the strong in vitro evidence highlighting the role of 4-hydroxybenzaldehyde in the GABAergic system, an underlying putative mechanism of action of G. elata against epilepsy (Ha et al., 2001, 2000; Tao et al., 2006), it is quite surprising, at least to the best of our knowledge, that the 4-hydroxybenzaldehyde has not yet been investigated concerning its anticonvulsant activity in acute seizure and/or epilepsy animal models. In fact, 4-hydroxybenzaldehyde showed an inhibitory activity against GABAT higher than that evidenced by HBA, the gastrodin metabolite (Choi and Lee, 2006; Ha et al., 2001). In addition, integrating all the aforementioned information on the anticonvulsant properties of G. elata extracts and/or its biocomponents, it seems clear that there is a lack of inclusion of positive controls (e.g. known AEDs) in the in vitro and in vivo assays using seizures/epilepsy models, which would be useful to better understand and clarify the anticonvulsant activity claimed for the biocomponents existing in C. elata and/or the several G. elata extracts.

Since the mechanisms of action of G. elata extracts and/or its constituents have been widely explored, they are briefly illustrated in Fig. 5. In this point, it is important to note that the two most important neurotransmitters involved in the regulation of brain neuronal activity are the excitatory neurotransmitter glutamate and the inhibitory neurotransmitter CABA. In fact, changes in the ratio of concentrations/activities of these compounds can contribute to increase or decrease the predisposition for seizures (Rowley et al., 2012). Therefore, several antiseizure mechanisms of action studied are oriented towards the involvement of these neurotransmitters, particularly the inhibition of enzymes involved in the degradation of GABA: GABA-T, SSADH and SSAR. In addition to the research works described in this review, although some other studies are not directly associated with anticonvulsant activity, they also demonstrated the involvement of glutamate and GABAergic systems in the action of several biocomponents present in G. elata. In this context, a study conducted by Jung et al. (2006) in mice demonstrated that 4-hydroxybenzaldehyde (100 mg/kg) can interfere with GABAergic nervous system (Fig. 5) because its effects were antagonized by flumazenil, a [GABA.sub.A] receptor antagonist (Jung et al., 2006). More recently, the NHBA analogue [N.sup.6]-(3-methoxy-4-hydroxybenzyl) adenine riboside (5 mg/kg) increased the GABA levels in hypothalamus (39%) and cortex (32%) and induced a significant decrease in glutamate contents in hypothalamus (22%) and cortex (21%) of ICR mice. The authors suggested that these effects could be associated with an increase in the glutamic acid decarboxylase enzyme activity in hypothalamus (43%) and cortex (31%) (Shi et al., 2014), which catalyses the decarboxylation of glutamate to GABA (Fig. 5). In addition, immunohistochemical studies also showed that NHBA (5 mg/kg, i.p.) increases the cFos expression in GABAergic neurons of the ventrolateral preoptic area of Sprague-Dawley rats, which suggests that NHBA activates the sleep centre in the anterior hypothalamus, producing significant sedative and hypnotic effects (Zhang et al., 2012).

Other molecular drug targets that also have a critical role in the CNS disorders, including in epilepsy, are the ion channels. Actually, the voltage-gated sodium channels are molecular targets for several commonly used AED (e.g. Phenytoin, carbamazepine and lamotrigine) as well as the calcium (e.g. gabapentin and ethosuximide) and potassium (e.g. retigabine) channels (Loscher et al., 2013). Nevertheless, data about the involvement of these ion channels in the putative anticonvulsant/antiepileptic mechanism of action of G. elata constituents remains scarce. In this regard, it was reported that gastrodin regulates the potassium and sodium voltage-gated ion channels in the small dorsal root ganglion neurons of diabetic rats (Sun et al., 2012) and also inhibited proton-gated currents mediated by acid-sensing ion channels in rat dorsal root ganglion neurons (Qiu et al., 2014).

The studies reported by Hsieh and collaborators (Hsieh et al., 2007, 2005) should also be highlighted because effectively go further in the research of potential epileptogenic processes than the classical models commonly used to study the mechanisms of seizures for the screening of new AED candidates. In fact, the complex mechanisms underlying the epileptogenesis are misunderstood, leading to a higher degree of difficulty concerning the emergence of new AEDs actually efficacious in the epilepsy progression, which are able to prevent refractory epilepsy as well as pharmacoresistance.

Since the AED candidates are intended to act in the CNS, it is usual to evaluate in parallel their neurotoxicity and anticonvulsant activity. In this context, the rotarod test seems to be the most used assay to infer about the neuronal toxicity during the screening stages of new AED candidates (Ghogare et al.. 2010; Hassan et al., 2012; Kumar et al., 2011). In the rotarod assay, the side effects in the CNS are manifested by the deficit in the motor coordination and this assay is usually used to perform an initial screening of neurotoxicity and, after, to calculate the median toxic dose ([TD.sub.50]), which allows the estimation of the protective index ([TD.sub.50]/[ED.sub.50]) of the compounds of interest. However, this test was not usually performed by the authors who had assessed the anticonvulsant activity of G. elata and/or its isolated constituents. An exception is the study conducted by Descamps et al. (2009), in which the HBA was evaluated to determine the minimal acute neurotoxicity through the rotarod assay, and at doses up to 200 mg/kg no neurological deficit, such as ataxia and sedation, was exhibited by C57 black J6 mice (Descamps et al., 2009).

Conclusion

The G. elata rhizome has been used as a traditional herbal medicine for centuries. Several in vitro and in vivo studies have been performed and many multi-pharmacological activities have been identified. However, the most promising activities exhibited by this medicinal herb and/or its phytochemical constituents are directed against several CNS disorders, such as epilepsy. Due to the relevance of this plant over the years, a more complete profile of the biochemical compounds isolated from G. elata has been made and the study of the pharmacokinetics of gastrodin (the most important phenolic compound) has brought an increased knowledge about its possible CNS actions. Moreover, several pharmacological mechanisms of action have been studied and proposed for the anticonvulsant activity of either G. elata extracts or its constituents and the available data appeared to be consistent and reproducible. However, further investigation is required on this field. Hence, more robust non-clinical studies and, in particular, clinical trials are required, not only to further investigate the anticonvulsant properties of G. elata extracts, but also to better understand what are the constituents involved in the described pharmacological actions, their mechanisms of action, toxicity profile and to confirm the claimed therapeutic activity against epilepsy.

Conflicts of interest

The authors have declared no conflicts of interest. Acknowledgments

The authors are grateful to Fundacao para a Ciencia e a Tecnologia (Lisbon, Portugal) for the PhD fellowship of Mariana Matias (SFHR/BD/85279/2012), involving the POPH-QREN, which is co-funded by FSE and MEC. The authors also acknowledge the support provided by FEDER funds through the POCI--COMPETE 2020 --Operational Programme Competitiveness and Internationalisation in Axis I--Strengthening research, technological development and innovation (Project No. 007491) and National Funds by FCT--Foundation for Science and Technology (Project UID/Multi/00709). The authors also acknowledge the contribution of Daniel Antunes Viegas for this review, particularly as English-speaking qualified person.

ARTICLE INFO

Article history:

Received 25 January 2016

Revised 29 August 2016

Accepted 3 September 2016

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Abbreviations: AED, antiepileptic drag; AUC, area under the concentration-time curve; [C.sub.max], peak plasma concentration; CNS, central nervous system: ED50, median effective dose; G. elata. Gastrodia elata; GABA-T. GABA transaminase; HBA. 4-hydroxylbenzyl alcohol; [IC.sub.50], median inhibitory concentration: i.v., intravenous; i.p., intraperitoneal; KA. kainic acid; LLOQ, lower limit of quantification; NHBA. [N.sup.6]-(4-hydroxybenzyl) adenine riboside; p.o., oral (per os); scPTZ, subcutaneous pentylenetetrazole; SSADH. succinic semialdehyde dehydrogenase: SSAR, succinic semialdehyde reductase: tip. elimination half-life; TDM, median toxic dose.

* Corresponding author at: Faculty of Health Sciences. University of Beira Interior, CICS-UBI--Health Sciences Research Centre. University of Beira Interior. Av. Infante D. Henrique, 6200-506 Covilha. Portugal. Fax: +351 275329099. E-mail address: gilberto@fcsaude.ubi.pt (G. Alves).

Mariana Matias (a,b), Samuel Silvestre (a,b), Amilcar Falcao (b,c), Gilberto Alves (a,b) *

(a) CICS-UBI--Health Sciences Research Centre, University of Beira Interior, Rua Marques d'Avila e Bolama, 6201-001 Covilha, Portugal

(b) CNC--Center for Neuroscience and Cell Biology, University of Coimbra, 3004-517 Coimbra, Portugal

(c) Department of Pharmacology, Faculty of Pharmacy, University of Coimbra, Polo das Ciencias da Saude, Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal

http://dx.doi.org/10.1016/j.phymed.2016.09.001

Table 1
Natural compounds isolated from G. elata.

Entry   Compound

The main bioactive compound

1       Gastrodin

General phenolic compounds

2       4-Hydroxybenzyl alcohol (HBA)
3       Vanillin
4       Vanillyl alcohol
5       4-Hydroxybenzaldehyde
6       4-Hydroxybenzylamine
7       4-Hydroxybenzyl methyl ether
8       Castrol
9       1,4-Benzenediol
10      Benzyl alcohol
11      Gastrodin A
12      Gastrol A
13      Gastrodin B
14      Gastrol B
15      4-Butoxyphenylmethanol
16      (4-Methoxyphenyl)methanol
17      Di-(4-hydroxybenzyl) hydroxylamine
18      4-Hydroxybenzyl ethyl ether
19      4-Hydroxybenzyl vanillyl ether
20      4-Hydroxybenzyl ether
21      Bis(4-hydroxybenzyl) ether mono-[beta]-D-glucopyranoside
22      4-([beta]-D-glucopyranosyloxy)benzyl alcohol
23      4-O-glucopyranosyl-benzaldehyde
24      2,4-Bis(4-hydroxybenzyl)phenol
25      4-Hydroxy-3-methoxybenzyl alcohol
26      4-Hydroxy-3-methoxybenzoic acid
27      4,4'-Dihydroxybenzyl sulfoxide
28      4-(4'-Hydroxybenzyloxy)benzyl methyl ether
29      Bis-(4-hydroxyphenyl) methane
30      Bis(4-hydroxybenzyl) sulfoxide
31      Bis(4-hydroxybenzyl) sulfide
32      5-(Hydroxymethyl)-2-furfuraldehyde
33      5-[4'-(4"-Hydroxybenzyl)-3'-hydroxybenzyloxyrnethyl]-furan-2-
          carbaldehyde
34      5-[4'-(4"-Hydroxybenzyl)-3'-hydroxybenzyl]-ftiran-2-
          carbaldehyde
35      5-(4-Hydroxybenzyloxymethyl)-furan-2-carbaldehyde
36      5-(Hydroxymethyl)-2-furaldehyde
37      5-((4-0-[beta]-D-glucopyranosylbenzyloxy)methyl)-furan-2-
          carbaldehyde
38      5-((4-0-[beta]-D-glucopyranosylbenzylsulfide)methyl)-furan-2-
          carbaldehyde
39      4-{{4-[4-(Methoxymethyl)phenoxy]benzyl)oxy}benzyl methyl
          ether
40      S-(4-hydroxybezyl)-glutathione
41      (-)-[gamma]-L-glutamyl-L-[S-(4-hydroxybenzyl)]
          cysteinylglycine
42      Methyl (-)-[gamma]-L-glutamyl-L-[S-(4-hydroxybenzyl)]
          cysteinylglycinate
43      (-)-([S.sub.s])-[gamma]-L-glutamyl-L-[S-(4-hydroxybenzyl)]
          cysteinylglycine sulfoxide
44      Ethyl (-)-([S.sub.s])-y-L-glutamyl-L-[S-(4-hydroxybenzyl)]
          cysteinylglycinate sulfoxide
45      (-)(-[R.sub.s]-[gamma]-L-glutamyl-L-[S-(4-hydroxybenzyl)]
          cysteinylglycine sulfoxide
46      Ethyl (-)-([R.sub.s])-[gamma]-L-glutamyl-L-
          [S-(4-hydroxybenzyl)]cysteinylglycinate sulfoxide
47      (-)-[gamma]-L-[N-(4-hydroxybenzyl)]glutamyl-L-
          [S-(4-hydroxybenzyl)] cysteinylglycine
48      (+)-L-[S-(4-hydroxybenzyl)]cysteinylglycine
49      (+MS)-[N-(4'-hydroxybenzyl)]pyroglutamate
50      (-)-(R)-[N-(4'-hydroxybenzyl)]pyroglutamate
51      Ethyl (+)-(S)-{N-[4'-hydroxy-3'-(4"-hydroxybenzyl)benzyl]}
          pyroglutamate
52      Ethyl (+)-(S)-2-hydroxy-3-[(4,-hydroxybenzyl)thio]propanoate
53      (+)-(S)-2-hydroxy-3-[(4'-hydroxybenzyl)thio]propanoic acid
54      Methyl (-)-(R)-2-hydroxy-4-[(4'-hydroxybenzyl)thio]butyrate
55      Methyl (+)-(S)-2-hydroxy-3-[(4,-hydroxybenzyl)thio]
          propanoate

Phenolic compounds containing a nucleoside

56      [N.sub.6]-(4-hydroxybenzyl)adenine riboside
57      [N.sub.2]-(p-hydroxybenzyl) guanosine

Phenolic conjugates containing a citrate moiety

58      Trimethylcitryl-[beta]-D-galactopyranoside
59      Parishin
60      Parishin B
61      Parishin C
62      Parishin D
63      Parishin E
64      Parishin F
65      Parishin G

Polysaccharides

66      AGEW
67      WTMA
68      WCEW
69      PGEB-3H

Other compounds

70      [beta]-Sitosterol
71      Sucrose
72      Trimethyl citrate
73      Uridine
74      Adenosine

Entry   Reference

The main bioactive compound

1       (Hsieh et al., 2007)

General phenolic compounds

2       (Hsieh et al., 2007)
3       (Hsieh et al., 2007)
4       (Hsieh et al., 2007)
5       (Hsieh et al., 2007)
6       (Tao et al., 2006)
7       (Taguchi et al., 1981)
8       (Hayashi et al., 2002)
9       (Huang et al., 2007)
10      (Lee et al., 2006)
11      (Li et al., 2007)
12      (Li et al., 2007)
13      (Zhang et al., 2013)
14      (Zhang et al., 2013)
15      (Ma et al., 2015)
16      (Ma et al., 2015)
17      (Hao et al., 1999)
18      (Yang et al., 2007)
19      (Han et al., 2011)
20      (Li et al., 2007)
21      (Taguchi et al., 1981)
22      (Taguchi et al., 1981)
23      (Ma et al., 2015)
24      (Li et al., 2007)
25      Gang et al., 2010)
26      (Lee et al., 2006)
27      (Li et al., 2007)
28      (Jang et al., 2010)
29      (Jang et al., 2010)
30      (Huang et al., 2007)
31      (Chen et al., 2015b)
32      (Huang et al., 2007)
33      (Huang et al., 2015b)

34      (Huang et al., 2015b)

35      (Huang et al., 2015b)
36      (Huang et al., 2015a)
37      (Li et al., 2016)

38      (Li et al., 2016)

39      (Han et al., 2011)

40      (Andersson et al., 1995)
41      (Guo et al., 2015a)

42      (Guo et al., 2015a)

43      (Guo et al., 2015a)

44      (Guo et al., 2015a)

45      (Guo et al., 2015a)

46      (Guo et al., 2015a)

47      (Guo et al., 2015a)

48      (Guo et al., 2015a)
49      (Guo et al., 2015b)
50      (Guo et al., 2015b)
51      (Guo et al., 2015b)

52      (Guo et al., 2015b)
53      (Guo et al., 2015b)
54      (Guo et al., 2015b)
55      (Guo et al., 2015b)

Phenolic compounds containing a nucleoside

56      (Huang et al., 2007)
57      (Wang et al., 2012)

Phenolic conjugates containing a citrate moiety

58      (Choi and Lee. 2006)
59      (Choi and Lee, 2006)
60      (Choi and Lee, 2006)
61      (Choi and Lee. 2006)
62      (Yang et al., 2007)
63      (Yang et al., 2007)
64      (Wang et al., 2012)
65      (Wang et al., 2012)
        (continued on next page)
Polysaccharides

66      (Chen et al., 2012)
67      (Chen et al., 2012)
68      (Chen et al., 2012)
69      (Ming et al., 2012)

Other compounds

70      (Shuchang et al., 2008)
71      (Shuchang et al., 2008)
72      (Huang et al., 2007)
73      (Huang et al., 2007)
74      (Huang et al., 2007)

Table 2
Correlation of the phytochemical constituents/extracts of C. elata
and their potential anticonvulsant activity, assessed in in vivo
studies.

Entry   Extract/bioactive     Animals (n)          Administration/
        component (dose)                           duration

1       Methanol extract      Male C57BL/6J        p.o., 12-12 h/2.5
          (500, 1000mg/ kg)     mouse (10-12)        days
2       Ether fraction of     Male ICR mouse.      p.o., daily, 14+4
          methanol extract      25-35 g(14)          days
          (200, 500 mg/kg)
3       Ether fraction of     Male                 p.o., daily, 10
          methanol extract      Sprague-Dawley       days
          (500mg/kg)            rat. 250-350 g
                                (30)
4       Ether fraction of     Male                 p.o., daily, 10
          methanol extract      Sprague-Dawley       days
          (500 mg/kg)           rat. 250-350 g
                                (30)
5       Aqueous extract       Male                 p.o., daily, 1
          (500, 1000 mg/kg)     Sprague-Dawley       week before KA
                                rat, 200-300 g
                                (3)
6       Aqueous extract       Male                 p.o., daily, 2
          (500, 1000 mg/kg)     Sprague-Dawley       weeks after KA
                                rat. 200-300 g
                                (3)
7       Ethanol extract       Male                 p.o., single dose
          (500, 1000 mg/kg)     Sprague-Dawley
                                rat, 200-300 g
                                (6)
8       Castrodin             Mongolian gerbil     p.o., daily, 1
          (60 mg/kg)            [100 (gastrodin      week
                                group) and 50
                                (control group)]
9       Castrodin             Male Kunming         i.p., single dose
          (25-100 mg/ml)        mouse, 28-33 g
                                (10)
10      Gastrodin             Male Kunming         i.p., single dose
          (25-100 mg/ml)        mouse, 28-33 g
                                (4-8)
11      Vanillyl alcohol      Male                 i.p., single dose
          (100, 200 mg/kg)      Sprague-Dawley
                                rat. 200-250 g
                                (6)

Entry   Control groups         Seizure/epilepsy      Brain parts
                               model

1       NT                     Cocaine               --
                                 (90 mg/kg, i.p)
2       NT                     KA (45 mg/kg, i.p.)   Hippocampus
3       NT                     PTZ (70 mg/kg,        --
                                 s.c., 3 days)
4       NT                     PTZ (25 mg/kg,        Postmitochondrial
                                 s.c., 8 weeks)        fraction
5       NT                     KA (12 mg/kg, i.p.)   Frontal cortex
          Valproic acid                                and hippocampus
          (250 mg/kg)
6       NT                     KA (12 mg/kg, i.p.)   Frontal cortex
          Valproic acid                                and hippocampus
          (250 mg/kg)
7       NT                     KA (2 [micro]g/2      Hippocampus
          N-nitro-L-arginine     [micro]l,
          methyl ester           stereotactic
                                 surgery)
8       NT                     Genetic seizure       Hippocampus
                                 model (seizure-
                                 sensitive
                                 gerbils)
9       NT Castrodin           Penicillin            --
          Phenytoin              (1000 000
                                 unit/ml)
10      NT Castrodin           Penicillin            Hippocampus
          Phenytoin              (560 000
                                 unit/ml)
11      NT Phenytoin           Ferric chloride       Bilateral
          (10 mg/kg. i.p)        (8 [micro]l,          cerebral
                                 intracortical         cortex
                                 injection)

Entry   Effects                                         Ref.

1       Delay cocaine-induced seizure onset             (Shin et al.,
        [down arrow] seizure duration (seconds)           2011)
        [up arrow] seizure latency (seconds)
2       500 mg/kg:                                      (Kimet al.,
        [up arrow] onset time of neurobehavioral          2001)
          changes
        [down arrow] seizure grade
        [up arrow] number of viable neurons (CA1 and
          CA3 regions)
3       [down arrow] recovery time (20.5 [+ or -]       (Ha et al.,
          3.2 min vs 35.0 [+ or -] 4.3 min of             2000)
          control)
        [down arrow] severity (2.8 [+ or -] 0.3 min
          vs 4.3 [+ or -] 0.4 min of control)
4       [up arrow] Brain CABA contents (2.0 [+ or -]    (Ha et al.,
          0.1 nmol/mg vs 1.2 [+ or -] 0.2 nmol/mg of      2000)
          untreated control)
5       [down arrow] counts of wet dog shakes           (Hsieh et al.,
        [down arrow] counts of paw tremor                 2007)
        [down arrow] counts of facial myoclonus
        [up arrow] phosphorylated JNK
        [up arrow] c-Jun protein
6       [down arrow] phosphorylated JNK                 (Hsieh et al.,
        [down arrow] c-Jun protein                        2007)
7       500 mg/kg:                                      (Hsieh et al.,
                                                          2005)
        [down arrow] neuronal NOS-staining cells
          (352.7 [+ or -] 118.0 cells vs 724.7
          [+ or -] 86.2 cells of KA group)
        1000 mg/kg:
        [down arrow] EDI-staining cells (332.5
          [+ or -] 180.0 cells vs 642.5 [+ or -]
          214.6 cells of untreated group)
        [down arrow] apoptotic cells (120.2 [+ or -]
          63.4 cells vs 542.5 [+ or -] 125.9 cells
          of untreated group)
        [down arrow] neuronal NOS-staining cells
          (219.0 [+ or -] 131.1 cells vs 724.7
          [+ or -] 86.2 cells of untreated group)
8       [down arrow] seizure score (average of 2.3)     (An et al.,
        [down arrow] GABA-T immunoreactivities            2003)
        [down arrow] SSADH immunoreactivities
        [down arrow] SSAR immunoreactivities
9       Phenytoin: gastrodin (1:50):                    (Zhou et al.,
        [up arrow] latent time of seizure (93.90          2015)
          [+ or -] 10.49 min vs 47.00 [+ or -] 11.72
          min of control)
        [up arrow] survival time (101.60 [+ or -]
          9.23 min vs 51.80 [+ or -] 11.14 min of
          the control)
10      Phenytoin: gastrodin (1:50):                    (Zhou et al.,
        [up arrow] percentage of spontaneous              2015)
          alternation
        [down arrow] latent time (9.59 [+ or -] 3.02
          s vs 48.26 [+ or -] 3.04 s Phenytoin group)
        [down arrow] fall-off in balance beam test vs
          Phenytoin group
        [up arrow] surviving neurons in CA1 area
          (173 vs 105 in non-treated group)
        [up arrow] surviving neurons in CA3 area
          (94 vs 44 in non-treated group)
        [up arrow] NF-[kappa]B relative density
          (0.86 [+ or -] 0.05 vs 0.23 [+ or -] 0.05
          of Phenytoin group)
11      [down arrow] wet dog shakes                     (Hsieh et al.,
        [down arrow] lipid peroxide levels                2000)
        200 mg/kg:
        [down arrow] luminol-chemiluminescence
          counts
        [down arrow] lucigenin-chemiluminescence
          counts

i.p., intraperitoneal: CABA. [gamma]-aminobutyric acid: CABA-T. CABA
transaminase; JNK. c-Jun N-terminal kinase: KA. kainic acid:
NF-[kappa]B. nuclear factor-[kappa]B; NOS. nitric oxide synthase: NT.
non-treatment: p.o., oral (per os); PTZ. pentylenetetrazole; s.c.,
subcutaneous: SSADH. succinic semialdehyde dehydrogenase: SSAR.
succinic semialdehyde reductase.

Table 3
Correlation of the phytochemical constituents and the methanol
extract of G. elata and their pharmacological activity, assessed in
in vitro studies.

        Extract/bioactive
        component
Entry   (concentration)              Assay

1       Methanol extract             MTT cell proliferation assay
          (0.1-30 [micro]g/ml)         (HT22 cell line)
                                     LDH assay
2       Gastrodin (400 [miro]M)      Enzyme assay for measurement of
                                       SSADH inhibition
3       HBA (10 [micro]g/ml)         Enzyme assay for measurement of
                                       GABA-T inhibition
4       4-Hydroxybenzaldehyde        Enzyme assay for measurement of
                                       GABA-T inhibition
                                     [[sup.3]H]Ro15-1788 competitive
                                       binding assay (benzodiazepine
                                       receptor)
                                     [[sup.3]H]flunitrazepam
                                       competitive binding assay
                                       (benzodiazepine receptor)
5       3,4-Dihydroxybenzaldehyde    Enzyme assay for measurement of
                                       GABA-T inhibition
                                     [[sup.3]H]Ro15-1788 competitive
                                       binding assay (benzodiazepine
                                       receptor)
                                     [[sup.3]H]flunitrazepam
                                       competitive binding assay
                                       (benzodiazepine receptor)
6       Vanillin                     Enzyme assay for measurement of
                                       GABA-T inhibition
                                     [[sup.3]H]Ro15-1788 competitive
                                       binding assay (benzodiazepine
                                       receptor)
                                     [[sup.3]H]flunitrazepam
                                       competitive binding assay
                                       (benzodiazepine receptor)
7       2,4-Dihydroxybenzaldehyde    Enzyme assay for measurement of
                                       GABA-T inhibition
                                     [[sup.3]H]Ro15-1788 competitive
                                       binding assay (benzodiazepine
                                       receptor)
                                     [[sup.3]H]flunitrazepam
                                       competitive binding assay
                                       (benzodiazepine receptor)
8       4-Hydroxybenzylamine         Enzyme assay for measurement of
          (10-50 [miro]M)              GABA-T inhibition
                                     Enzyme assay for measurement of
                                       SSADH inhibition
9       4-Hydroxybenzaldehyde        Enzyme assay for measurement of
          (10-25 [miro]M)              GABA-T inhibition
                                     Enzyme assay for measurement of
                                       SSADH inhibition
10      Vanillin (100 [miro]M)       Measurement of intracellular
                                       [Ca.sup.2+] (IMR-32 human
                                       neuroblastoma cell line)
                                     Flow cytometry (IMR-32 human
                                       neuroblastoma cell line)
11      4-Hydroxybenzaldehyde        Measurement of intracellular
          (100 [micro]M)               [Ca.sup.2+] (IMR-32 human
                                       neuroblastoma cell line)
                                     Flow cytometry (IMR-32 human
                                       neuroblastoma cell line)
12      Trimethylcitryl-[beta]-D-    Enzyme assay for measurement of
          galactopyranoside            GABA-T inhibition
          (10 [micro]g/ml)
13      S-(4-                        [[sup.3]H]kainic acid binding
          hydroxybenzyljglutathione    assay (glutamate receptor)

Entry   Main results                                    Reference

1       [up arrow] cell viability                       (Han et al.,
                                                          2014)
        [down arrow]. LDH release (69.2% for 1
          [micro]g/ml and 66.5% for 5 [micro]g/ml)
2       15% remaining activity                          (Baek et al.,
                                                          1999)
3       30.87 [+ or -] 3.06% vs 65.38 [+ or -] 1.45%    (Choi and Lee,
          (valproic acid)                                 2006)
4       [IC.sub.50]=4.13 [micro]g/ml vs [IC.sub.50] =   (Ha et al.,
          132.89 [micro]g/ml (vigabatrin)                 2001)
        90%. 90% and 92% ([10.sup.-8] M, [10.sup.-6]
          M and [10.sup.-4] M, respectively)
        55%. 54% and 55% ([10.sup.-8] M, [10.sup.-6]
          M and [10.sup.-4] M, respectively)
5       [IC.sub.50]=4.78 [micro]g/ml vs [IC.sub.50] =   (Ha et al.,
          132.89 [micro] g/ml (vigabatrin)                2001)
        118%. 118% and 116% ([10.sup.-8] M,
          [10.sup.-6] M and [10.sup.-4] M,
          respectively)
        91%. 86% and 85% ([10.sup.-8] M, [10.sup.-6]
          M and [10.sup.-4] M. respectively)
6       1CS0 = 5.37 [micro]g/ml vs [IC.sub.50] =        (Ha et al.,
          132.89 [micro]g/ml (vigabatrin)                 2001)
        75%, 68% and 68% ([10.sup.-8] M, [10.sup.-6]
          M and [10.sup.-4] M, respectively)
        54%. 58% and 58% ([10.sup.-8] M. [10.sup.-6]
          M and [10.sup.-4] M, respectively)
7       [IC.sub.50]=7.80 [micro]g/ml vs [IC.sub.50] =   (Ha et al.,
          132.89 [micro]g/ml (vigabatrin)                 2001)
        Similar to 3,4-dihydroxybenzaldehyde
        Similar to 3,4-dihydroxybenzaldehyde
8       [IC.sub.50] = 15.4 [micro]M                     (Tao et al.,
                                                          2006)
        [IC.sub.50] > 1000 [micro]M
9       [IC.sub.50] = 16.5 [micro]M                     (Tao et al.,
                                                          2006)
        [IC.sub.50] = 24.7 [micro]M
10      [down arrow] relative % of glutamate-induced    (Lee et al.,
          intracellular [Ca.sup.2+]                       1999)
        [down arrow] relative % of glutamate-induced
          apoptosis
11      [down arrow] relative % of glutamate-induced    (Lee et al.,
          intracellular [Ca.sup.2+]                       1999)
        [down arrow] relative % of glutamate-induced
          apoptosis
12      56.80 [+ or -] 0.21% vs 65.38 [+ or -]1.45%     (Choi and Lee,
          (valproic acid)                                 2006)
13      [IC.sub.50] = 2 x [10.sup.-6] M vs 2 x          (Andersson et
          [10.sup.-7] M (glutamate) and 8 x               al., 1995)
          [10.sup.-7] M (glutathione)

[Ca.sup.2+], calcium; GABA-T, [gamma]-aminobutyric acid transaminase;
HBA. 4-hydroxylbenzyl alcohol; [IC.sub.50], median inhibitory
concentration; MTT. 3-[4.5-dimethylthiazol-2-yl]-2.5-
diphenyltetrazolium bromide; SSADH, succinic semialdehyde
dehydrogenase.


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Author:Matias, Mariana; Silvestre, Samuel; Falcao, Amilcar; Alves, Gilberto
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:4EUPR
Date:Nov 15, 2016
Words:14796
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