Exploration of the Protective Effects of Some Natural Compounds against Neurodegeneration Exploiting Glycine Receptors in vivo Model

The commencement of the presented project was by screening of natural compounds extracted from neuroactive endogenous medicinal plants. Explicitly, ferutinin (Ferula hermonis L.), thymoquinone (Nigella sativa L.), eugenol (Syzygium aromaticum L.) and 6-gingerol (Zingiber officinale L.) were tested for their protective effect against neurodegeneration utilizing Glycine receptors (GlyRs) in vivo model. None of these compounds were reported before to modulate the in vivo GlyRs. GlyRs are inhibitory key mediators of synaptic signaling in spinal cord, brain stem, and higher central nervous system regions. Neurodegeneration may cause alteration of the GlyRs causing strychnine-like convulsions and stiffness. Modulation of GlyRs in vivo was studied in a mouse model of strychnine toxicity. Ferutinin revealed to be potent modulators to GlyR; with potential anticonvulsant properties in low doses. Thymoquinone, eugenol and 6-gingerol when given together with strychnine, in low concentrations reduce strychnine toxicity by reversing strychnine toxicity in mice. It could be concluded that all compounds under investigation could be used as sedatives in low doses. In order to fight against neurodegenerative diseases is to improve body antioxidant, the compounds under investigation provided to be good sources for antioxidant potential. In brief, ferutinin, thymoquinone, eugenol and 6-gingerol suggested being novel GlyR modulators, good phytochemicals, pharmacological tool and a dose sensitive drug to treat stiffness, convulsions and prophylactic agents to guard against neurodegenerative disorders. *Corresponding author: Raafat KM, Department of Pharmaceutical Sciences, Faculty of Pharmacy, Beirut Arab University, 115020 Beirut, Lebanon, Tel: +96176014620; E-mail: k.raafat@bau.edu.lb Received July 25, 2013; Accepted August 12, 2013; Published August 19, 2013 Citation: Raafat KM (2013) Exploration of the Protective Effects of Some Natural Compounds against Neurodegeneration Exploiting Glycine Receptors in vivo Model. Nat Prod Chem Res 1: 113. doi:10.4172/ 2329-6836.1000113 Copyright: © 2013 Raafat KM. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


Introduction
The use of endogenous natural products with therapeutic properties is as ancient as human civilization and, for a long time, plant products were the main sources of drugs [1]. In an attempt to find potential neuroprotective agents from plants against neurodegeneration, we examined whether a number of natural products of endogenous herbal medicines may exhibit protective efficacy on the in vivo GlyRs.
The selection of the natural compounds under investigation was based on screening of many neuroactive compounds, utilizing preliminary electrophysiological studies using patch-clamp. Therefore, the choice of quinone and phenolic compounds under investigation was based on their preliminary modulatory effects in the in vitro homomeric α1 GlyR transfected on HEK 293 cells.
There is increasing evidence for many potential benefits through quinone and phenolic compounds mediated regulation of cellular processes such as inflammation and neuroactivity [2]. Inductive or signaling properties of quinones and phenolics may occur at concentrations much lower than required for effective radical scavenging [2]. Moreover, quinones and phenolics demonstrate numerous biological and pharmacological effects, including antiinflammatory, and anticarcinogenic [3][4][5].
Sesquiterpene compounds exhibit numerous biological and pharmacological effects, including antifungal and phytoestrogen [6], as well as apoptotic effect [7]. Several species of Ferula genus has been used in folk medicine in digestive disorders, rheumatism, headache, arthritis and as, antispasmodic and aphrodisiac [8]. Ferutinin (Jaeschkeanadiol p-hydroxy benzoate) ( Figure 1A) is mainly found in Ferula hermonis L. "Zallouh", and other Ferula species (like, Ferula tenuisecta) [9]. It belongs to a broad group of sesquiterpene ducane esters [10] and has a wide range of biological activities. These include neuro-pharmacological actions such as tranquilizers and mild sedation. Ferutinin was found to stimulate nitric oxide synthase activity in median eminence of the rat [8].
Nigella sativa L. seeds contain 0.4%-2.5% essential oil. The major component was the quinone, thymoquinone (27.8%-57.0%) ( Figure  1B) [11]. Most properties of whole seeds or their extracts are mainly attributed to quinone constituents [12]. More recently, a great deal of attention has been given to this pharmacologically active quinone. It has been shown that thymoquinone has several properties including analgesic and anti-inflammatory actions [13,14], protection against chemicals induced carcinogenesis [15], the inhibition of membrane lipid peroxidation [14], anticonvulsant activity in the petit mal epilepsy probably through an opioid receptor-mediated increase in GABAergic tone [11].
Eugenol (4-allyl-2-methoxyphenol) ( Figure 1C), a phenylpropanoid compound, is a major constituent of Syzygium aromaticum [16]. Eugenol has anticonvulsive and hypothermic activities. It also has antioxidant properties and protects neurons in culture from toxic events. Eugenol is also widely used as an analgesic in dentistry [17]. 6-Gingerol is a pungent principle in the rhizome of ginger and possesses the labile β-hydroxy keto functional group, which makes it susceptible to transformation to less-pungent compounds such as shogaols and zingerone by elevated temperature. 6-Gingerol has been reported to exhibit many interesting pharmacological and physiological functions for example; antipyretic, cardiotonic, chemopreventive, anti-inflammatory and antioxidant properties [18,19]. However, the neuroprotective potential of 6-gingerol is still under investigation and needs to be clarified. None of these compounds were tested on the in vivo GlyRs.
In our previous work, it was proved that phenolics in vivo and in vitro have potent modulatory actions on GlyRs [20,21]. The inhibitory GlyRs is a member of the cysteine loop superfamily of ligand-gated ion channel receptors. It shares structural similarity with nicotinic acetylcholine [22]. The GlyRs is chiefly expressed in spinal cord, brain stem, and other regions of the central nervous system, where it mediates rapid synaptic inhibition [22][23][24][25][26]. Strychnine possesses convulsion action due to interference with postsynaptic inhibition mediated by glycine. Glycine is a key inhibitory transmitter to motor neurons and interneurons in the spinal cord, and strychnine acts as a selective, competitive antagonist to block the inhibitory effects of glycine at all glycine receptors [20]. Neurodegeneration can cause alteration of the GlyRs causing strychnine-like convulsions and stiffness [27]. Flavonoids, alcohols and local anesthetics have been shown to modulate GlyRs function [20]. In this study, the aim of combination of natural compounds under investigation with strychnine ( Figure 1D and 1E), is to explore the effect of these extracts and phenolics on neurodegeneration, taking GlyRs as in vivo model [20,27]. Such combinations were used before in traditional medicine [20,28,29]. Nevertheless, all natural compounds under investigation were not reported to modulate the in vivo GlyRs. The combined effects of the quinone or phenolic compounds on strychnine lethality may be synergistic or antagonistic. Accordingly, we investigated the interaction of these substances via in vivo strychnine lethality test. The aim of this study was to examine the protective effect of ferutinin, thymoquinone, eugenol and 6-gingerol against neurodegeneration by the manipulation of the in vivo GlyRs and finding their radical scavenging potential.

Sample preparation
Ferula hermonis roots and seeds, Nigella sativa seeds, Syzygium aromaticum flower-buds and Zingiber officinale rhizomes were separately dry ground using TCM grinder (TCM, China). All fine powders were extracted using hexane, ethyl acetate and 80% ethanol respectively, and were stirred for 24 hours in their liquors. During which the flasks were covered by aluminum foil to prevent the light damage. After 24 hours, the extracts were double filtered through a porcelain funnel using 20-25 μM filters. The filtered extracts were well dried using Rotavap (Buchi, Germany) at temperature 40°C under vacuum.

Reagents and chemicals
Reference Standard ferutinin, thymoquinone, eugenol, 6-gingerol, strychnine and HPLC solvents were commercially purchased (Sigma-Aldrich, Germany). All standards were dissolved in dimethyl sulphoxide (DMSO) for the in vivo test, and in methanol in the radical scavenging experiment.

Fractionation and isolation of the effective compound
All extracts were fractionated using column chromatography (CC). Preparative chromatography column was used. Gradient elution was employed utilizing normal phase silica gel as stationary phase. During the entire chromatography process the eluent was collected in a series of fractions by time. Fractions were analyzed using TLC and RP-HPLC compared to commercial standards. HPLC analysis was carried out in a JASCO instrument (JASCO, Japan). Solvents were degassed by an online degasser of the ProStar System. The column used was a RP C18 endcapped Lichrospher column (250×4.6 mm I.D.; 5 μM particle size) was employed (Merck, Darmstadt, Germany), at 30°C. The injection volume was 20 μL and UV detection was performed using UV detector tested using JASCO spectrophotometer (JASCO, Japan) ( Figure 10).

Behavioral studies
Male Swiss-Webster mice (Faculty of Pharmacy, Beirut Arab University (BAU), Beirut, Lebanon) were housed for 1 week prior to experimentation. The environment consisted of standard mouse cages with a 12-h light/dark cycle. The temperature was 22 ± 1°C, animals had free access to water and standard laboratory pellets (20% proteins, 5% fats, and 1% multivitamins [20]. All animal care and experiments were done in accordance to Beirut Arab University Institutional Animal Care & Use Committee and with approval of the local ethics commission. All data were tested for significance using one-way ANOVA. A value of p<0.05 was considered statistically significant.

Tonic extensor convulsion and toxicity tests
Male mice with an average weight of 18-22 g were used. Reference mice received a single ip injection of 2 mg/kg strychnine to determine the pharmacological end point for tonic extensor convulsions (TECs) and lethality. Control mice were injected ip with 0.2 ml Dimethyl sulfoxide (DMSO) followed 30 minutes later by an ip injection of 2 mg/kg strychnine in DMSO [20]. Test mice were injected ip with serial dilutions of ferutinin, thymoquinone, eugenol or 6-gingerol, and 30 min. later injected ip with 2 mg/kg strychnine. Mice were observed over a period of 1 hour and the time until occurrence of TECs and death was recorded. The first presence of tonus in the hind limbs with stretching was taken as TEC onset, whereas death, preceded by clonic convulsions and tonic seizures, was the pharmacological end point. Another control group of mice was injected with ferutinin, thymoquinone, eugenol or 6-gingerol only; in the highest doses, neither of the test compounds (except strychnine) alone produced convulsions or toxicity.

Results and Discussions
Quinone and phenolic compounds under investigation, namely, ferutinin, thymoquinone, eugenol and 6-gingerol ( Figure 1) have shown positive modulation to GlyRs utilizing preliminary electrophysiological studies using patch-clamp. The protective effects of these compounds against neurodegeneration were mainly investigated in this study using in vivo behavioral studies on mice and radical scavenging activity on DPPH.

TEC and lethality test
When administered alone, ferutinin, thymoquinone, eugenol and 6-gingerol at their highest concentrations did not produce any convulsions. None of the tested animals showed any TECs, and test compounds (except strychnine) alone were not otherwise toxic even in highest concentration tested or lethal in any of the test animals. In contrast, an injection of strychnine alone (2 mg/kg ip) was lethal in all tested animals ( Figure 2). TEC set in after 7.85 ± 0.35 minutes and death occurred after 8.5 ± 0.42 minutes (n=4). In mice that had been preinjected with either ferutinin (0.1, 0.5 and 1 mg/kg), thymoquinone (1, 3 and 5 mg/kg), eugenol (0.1, 1 and 10 ml/kg) or 6-gingerol (0.1, 1 and 10 mg/kg ip).
In this study, we investigated the in vivo effects of ferutinin isolated by column chromatography from seeds of Ferula hermonis (isolated ferutinin) and that of the standard ferutinin ( Figure 1A). The purity of the isolated ferutinin was about 96% measured using standard ferutinin calibration curve and spectrophotometer absorbance at 254 nm. In mice that had been preinjected with standard ferutinin (0.1 mg/kg), the toxic effects of strychnine were almost reversed. As, mice featured slight and short reversible TEC without any lethal effect for more than an hour (Figure 2). On the other hand, better effects were shown with isolated ferutinin. As, the mice that had been preinjected with the isolated ferutinin (0.1 mg/kg), the toxic effects of strychnine were completely reversed. The mice were completely protected, as they did not feature neither TEC nor lethal effect for more than an hour ( Figure 2). Standard ferutinin at (0.5 mg/kg) significantly counter acted the strychnine toxicity and TEC happened at 9.4 ± 0.9 minutes and death occurred after 10.03 ± 0.8 minutes (n = 4) ( Figure 2). While, isolated ferutinin (0.5 mg/kg ip) did not significantly protect the mice from strychnine toxicity. Nevertheless, the highest dose of standard ferutinin (1 mg/kg ip) did not significantly protect the mice from strychnine toxicity. While, the highest dose of isolated ferutinin (1 mg/ kg ip) slightly aggravated strychnine toxicity.
This biphasic effect of ferutinin, protective in low doses and aggravating strychnine toxicity in high doses, towards GlyR were observed before in elevation of intracellular calcium in leukemia human Jukart T-cell line [7] and Zn 2+ ions on the strychnine-sensitive glycine receptor [30]. Therefore, it could be concluded that ferutinin alone, in low doses (0.1 mg/kg ip) protected the mice from strychnine lethality, as expected from GlyR potentiator.
The isolated Tq purity was about 89% measured using standard Tq calibration curve and spectrophotometer absorbance at 254 nm. The toxic effects of strychnine were significantly reduced to about 3 folds (thymoquinone protective effect against TEC = 66.67 ± 0.50% and against lethality 40.10 ± 0.90%) (Table 1) in the relatively low concentrations (1 mg/kg ip) of thymoquinone ( Figure 3). TEC was slightly aggravated, while the toxicity slightly decreased with increasing the concentration of thymoquinone (3 mg/kg ip). Thymoquinone (5 mg/kg ip) showed slight TEC aggravation and non-significant change in toxicity compared to solvent treated group.
The purity of the isolated EUG was about 84% measured using standard ferutinin calibration curve and spectrophotometer absorbance at 215 nm. Strychnine toxic effects were significantly reduced to about 2 folds in the relatively low concentrations (0.1 ml/kg ip) of eugenol

STR-TEC Protective Capacity (in vivo)
at Lowest Concentration (%) and against lethality 56.04 ± 0.81 %) ( Table 1). The reduction of TEC and toxicity was slightly increased with increasing the concentration of eugenol (1 ml/kg ip). Eugenol (10 ml/kg ip) showed about 3 folds reduction in TEC and lethality of strychnine.

Ft
The isolated 6-Gn purity was about 90% measured using standard 6-Gn calibration curve and spectrophotometer absorbance at 282 nm. 6-Gingerol (0.1 ml/kg ip) showed slight protection against strychnine TEC and toxicity (6-gingerol protective effect against TEC = 9.86 ± 0.86 % and against lethality 21.97 ± 0.97%) ( Table 1). TEC was slightly aggravated, while the toxicity not significantly changed with increasing the concentration of 6-gingerol (1 ml/kg ip). 6-Gingerol (10 ml/kg) showed aggravation of strychnine TEC and lethality. Therefore, as expected form GlyRs modulators, it could be concluded that ferutinin, thymoquinone, eugenol and 6-gingerol when given together with strychnine, in low concentrations reduce strychnine toxicity. It could be concluded that all compounds under investigation could be used as sedatives in low doses as they potentiate the inhibitory GlyRs.
In vivo data showed that ferutinin has a biphasic effect. At low doses (0.1 mg/kg ip in vivo), potentiates the GlyRs. While, in relatively high doses (1 mg/kg ip in vivo) inhibits the GlyRs. In low concentrations, ferutinin showed to be the most potent protective agent against strychnine toxicity, followed by thymoquinone and eugenol. While, 6-gingerol was the least potent protective agent.

Quinone and phenolic compounds radical scavenging activity on DPPH
In order to fight against neurodegenerative diseases is to improve body antioxidant, the compounds under investigation provided to be good sources for antioxidant potential. A concentration-dependent assay was carried out with ferutinin, thymoquinone, eugenol and 6-gingerol, and the results are presented in Figures 5-9 respectively. These results provide a direct comparison of the antioxidant activity with ascorbic acid (AA). Ferutinin, thymoquinone, eugenol and 6-gingerol possessed significant scavenging activity on the DPPH radical and acted as an antioxidant. The scavenging effect was increased with increasing concentration and reaction time (Figures 5-9).

Conclusion
We have presented scientific evidence that natural products extracted from neuroactive endogenous medicinal plants, namely, ferutinin, thymoquinone, eugenol and 6-gingerol were verified to be possible modulators to GlyRs; with potential anticonvulsant properties in low doses. These compounds proved to be potential scavengers fighting against neurodegenerative diseases by improving body antioxidant potential. The quinone and phenolic compounds under investigation is suggested to be prophylactic agents acutely administered in response to GlyRs strychnine intoxication and can prevent toxic symptoms, including seizures, convulsions and death.