Anticonvulsant and Antioxidant Effects of Tilia americana var. mexicana and Flavonoids Constituents in the Pentylenetetrazole-Induced Seizures

Tilia genus is commonly used around the world for its central nervous system properties; it is prepared as tea and used as tranquilizing, anticonvulsant, and analgesic. In this study, anticonvulsant activity of the Tilia americana var. mexicana inflorescences and leaves was investigated by evaluating organic and aqueous extracts (100, 300, and 600 mg/kg, i.p.) and some flavonoids in the pentylenetetrazole-induced seizures in mice. Moreover, antioxidant effect of these extracts and flavonoids was examined in an in vitro study by using spectrophotometric technique. Significant activity was observed in the methanol extract from inflorescences. An HPLC analysis of the methanol extract from inflorescences and leaves of Tilia allowed demonstrating the respective presence of some partial responsible flavonoid constituents: quercetin (20.09 ± 1.20 μg/mg and 3.39 ± 0.10 μg/mg), rutin (3.52 ± 0.21 μg/mg and 8.94 ± 0.45 μg/mg), and isoquercitrin (1.74 ± 0.01 μg/mg and 1.24 ± 0.13 μg/mg). In addition, significant but different antioxidant properties were obtained among the flavonoids and the extracts investigated. Our results provide evidence of the anticonvulsant activity of Tilia reinforcing its utility for central nervous system diseases whose mechanism of action might involve partial antioxidant effects due to the presence of flavonoids.


Introduction
The Tilia genus belongs to the Tiliaceae family, which consists of 25-80 species, and it is mainly distributed in Europe and Asia, with a few species in North America [1,2]. In México, T. americana is known as Tilia and it is distributed in 14 states across both the northern and southern parts of the country [3]. This plant has a wide geographical distribution; 2 Oxidative Medicine and Cellular Longevity esters, aliphatic acids, polyphenols, and terpenoids [10][11][12] with demonstrated heterogeneity in their flavonoid patterns when flowers and leaves are sampled independently [13][14][15]. Nevertheless, the knowledge of the potential contribution of the differences in the flavonoid pattern between inflorescences and leaves of Tilia on its therapeutic properties is unknown.
In previous studies, significant and dose-dependent antinociceptive and anxiolytic-like activities were demonstrated after administration of hexane, methanol, and aqueous extracts of Tilia inflorescences (10-300 mg/kg), where glycosides of quercetin and kaempferol were characterized as the main active compounds of the inflorescences [7,8,16].
Although, it has been demonstrated that these metabolites might facilitate the inhibitory response of the central nervous system (CNS), due to modulating the GABAergic and serotoninergic systems [17][18][19], it is unknown if these are the only possible mechanisms of action. The aim of this study was to investigate if Tilia organic and aqueous extracts and some of its flavonoids are able to control seizure activity in mice receiving a pentylenetetrazol (PTZ, GABA antagonist) [18,19] and whether these extracts possess antioxidant properties.

Drugs.
Kaempferol, quercetin, rutin, astragalin, isoquercitrin, and quercitrin standards, as well as PTZ, tween 80, and diazepam, were purchased from Sigma (Sigma-Aldrich Co., St Louis, MO, USA). Extracts were resuspended in saline solution (s.s. 0.9%, NaCl) alone or in 0.2% tween 80 in s.s. Control animals received the same volume of the vehicle via the same administration route. PTZ and flavonoids were dissolved in s.s. and diazepam in 0.2% tween 80 in s.s. Drugs were freshly prepared on the day of the experiments. All treatments were injected using an intraperitoneal (i.p.) administration within a volume of 10 mL/kg body weight.

Preparation of the Plant Extract.
For the preparation of the organic extracts of Tilia, the air-dried powdered inflorescences (1.8 kg) were successively extracted with hexane (4 L × 3) and methanol (4 L × 3) by maceration at room temperature (22 ∘ C). Regarding the leaves, 317 g was successively extracted with hexane (1 L × 3) and methanol (1 L × 3) by maceration at room temperature (22 ∘ C). The solvents were separated from the residues by gravity filtration and then evaporated in vacuum. The final crude extracts were obtained in percentage from dry weight (% d.w.) as follows: (a) for inflorescences 25 g of a yellow greasy hexane extract (1.4% d.w.) and 130 g of a dark brown syrupy methanol extract (7.2% d.w.), (b) whereas, in the case of the leaves (317 g), the yield was hexane 3.0 g (0.95%) and 29.8 g (9.4%) methanol crude extracts.
In the case of the aqueous extracts of Tilia inflorescences, the air-dried powdered inflorescences (24 g) were extracted by infusion in boiling water (500 mL) for 10 min. The resulting extract was separated from its residue by gravity filtration; samples were frozen in liquid nitrogen and freeze-dried for 12 h in a Heto FD3 Lab lyophilizer. The final crude aqueous extract consisted of 1.3 g of a yellow powder or 5.3% from dry weight, whereas, in the aqueous extract from the leaves (26 g), 3.58 g of extract or 13.76% from dry weight was obtained.

Animals.
Female Swiss albino mice (25-30 g, National Institute of Psychiatry Ramon de la Fuente Muñiz) were used for the in pharmacological tests. The animals were kept at a constant room temperature (22 ∘ C) and maintained in a 12 h/12 h light/dark cycle. All experimental procedures were carried out according to a protocol approved by the local Animal Ethics Committee (NC093280.2) in compliance with national (NOM-062-ZOO-1999) and international regulations on the care and use of laboratory animals. The animals were fed ad libitum with standard feed and water during the course of the study.

Pharmacological
Evaluation. All mice were treated through a daily s.s. injection for 5 days before the treatments were administered. For each experimental procedure, animal groups consisted of six mice. Diazepam was used as a reference drug. The extracts were evaluated at doses of 100, 300, and 600 mg/kg, i.p. (these dosages were taken into account from references of the literature), whereas flavonoids were tested at a dosage established depending on their presence in the active extract. Treatments were evaluated 30 min (reference and flavonoids) and 60 min (extracts) after administration.
Given that only methanol and aqueous extracts showed significant activity, it was interesting to analyze the possible anticonvulsant effect of these corresponding leave extracts at a dosage of 600 mg/kg. In the case of identified flavonoids, given that synergism among the constituents might be involved in the effect of the crude extract, it was decided to test the individual flavonoids in dosage approximate 10fold more than that found in the crude extract. Control animals received the vehicle by the same route and volume of administration.

High Performance Liquid Chromatography (HPLC)
Analysis. A sample (1 mg) of each methanol extract was dissolved in 1 mL of methanol and filtered to inject 20 L to the HPLC system using an Agilent Technologies Chromatograph, ODS hypersil C-18 column (125 mm × 4 mm i.d., 5 m particle size), and UV detector (Agilent, G1365BMWD, multiwavelength detector). The system was run with an isocratic gradient of acetonitrile-water 15 : 85; water was acidulated with trifluoroacetic acid to reach 2.5 pH. The column was maintained at 30 ∘ C with a flow of 1 mL/min. Total running time was 14 min. The calibration curve was based on standards of previously reported flavonoids of Tilia species such as quercetin, rutin, and isoquercitrin ( Figure 1). Chromatograms were detected at 350 nm. •− in the assay system and xanthine oxidase activity were measured by an NBT reduction using a DU-640 series Beckman spectrophotometer. This system can be used to test the O 2 •− scavenging capacity only when the samples do not interfere with xanthine oxidase activity. A compound with O 2 •− scavenging capacity should decrease the NBT reduction without interfering with the xanthine oxidase activity measured as uric acid production. A total of 800 L of the following reaction mixture was mixed with 100 L of different concentrations of Tilia extracts: 90 M xanthine, 16 mM Na 2 CO 3 , 22.8 M NBT, and 18 mM phosphate buffer (pH 7.0). The reaction was started by the addition of 100 L of xanthine oxidase (168 U/L). The optical density was recorded at 295 nm for uric acid production and 560 nm for O 2 •− in the assay system [15]. The scavenging percentage was obtained from the optical density at 560 nm. NDGA (0, 1, 2, 3.02, and 8 g/mL) was used as a standard for O 2 •− scavenging in this assay.

Hypochlorous Acid (HOCl) Scavenging Capacity.
The catalase assay involved the spectral analysis of the enzyme. We generated samples for spectra of catalase (200-500 nm), catalase treated with HOCl, and catalase containing varied mixtures of HOCl treated with increasing concentrations of Tilia extracts and a reference compound. The HOCl scavenging capacity of these extracts or the reference antioxidant was evident by the inability of HOCl to eliminate/decrease in a concentration-dependent manner. Experiments were carried out essentially as described previously [24].
A solution of 49.8 M bovine liver catalase (16.6 M, final concentration) was mixed with 18 mM HOCl (6 mM, final concentration) in the presence of increasing concentrations of Tilia extracts or the reference compound of ascorbic acid (0, 22, 44, 88, and 176 g/mL). Spectra (370-450 nm) of catalase alone, catalase plus HOCl, or catalase plus HOCl and the Tilia extracts or the reference compound were generated, and the optical density (OD) at 404 nm was recorded. The OD of catalase alone minus the OD of catalase plus HOCl was considered to be 100% of catalase degradation (or 0% of scavenging activity).
The difference in the OD of the catalase alone minus the OD of the catalase plus HOCl in presence of either extracts or the reference compound was compared with this value and the ability of these to scavenge HOCl was compared with that of ascorbic acid. were dispensed in 1.5 mL Eppendorf tubes and mixed with 10 L of HPLC-grade methanol immediately followed by the addition of 0.9 mL of FOX reagent. Samples were then vortexed for 5 s and incubated at room temperature for 10 min. The tubes were centrifuged at 15,000 ×g for 10 min, and the absorbance at 560 nm was read against a methanol blank.   (Figure 2(c)). Regarding leaves of Tilia, the methanol and aqueous extracts did not modify the onset of seizures at the highest dosage tested (600 mg/kg, i.p.) (Figures 2(a)-2(c)).

The Anticonvulsants Effects on PTZ-Induced
The percentage of animals showing myoclonic, clonictonic, and tonus behavior in the control group was 100%. By other side, a decreased in the percentage of mice showing tonic seizures was observed in the three tested inflorescence extracts as follows: (a) in hexane extract at 300 mg/kg (33%) and 600 mg/kg (16%); (b) in methanol extract at 300 mg/kg (33%) and 600 mg/kg (83%); (c) in aqueous extract at 100 mg/kg (16%), 300 mg/kg (50%); and 600 mg/kg (83%). Regarding the leaves extract, a reduction was observed in the presence of tonic seizures after methanol (16%) and aqueous (33%) extracts.
In reference to the flavonoids, the aglycone quercetin (100 mg/kg, i.p.) did not modify neither latency nor percentage of seizures in mice, whereas its glucosides rutin (quercetin-3-O-rutinoside) and isoquercitrin (quercetin-3-O--D-glucopyranoside) significantly delayed the presence of the first tonic seizure and reduced the percentage of mice showing this kind of convulsion (Table 1).

HPLC Analysis from Flowers and Leafs of Tilia americana var. mexicana.
According to the concentration of flavonoids determined from three peaks in the HPLC analysis ( Figure 3, Table 2), the presence of quercetin (aglycone), rutin, and isoquercitrin glycosides expected in the highest dosage (600 mg/kg) of methanol extracts from inflorescences and leaves administered in mice would be as follows: regarding inflorescences 12 mg of quercetin (Figure 3(a)), 2.11 mg of rutin (Figure 3(b)), and 1.04 mg of isoquercitrin (Figure 3(c)), but in the leaves 0.54 mg of quercetin, 5.36 mg of rutin, and 0.74 mg of isoquercitrin.

Antioxidant Effects on Free Radical
Scavenging. All the Tilia extracts successfully scavenged the reactive species studied. The analysis of the IC 50 values indicated that both Data are represented as the mean ± SE of three independent assays. * < 0.05, ANOVA followed by Tukey's test. tested methanol and hexane extracts of Tilia (leaves and inflorescences) display scavenging activity in a concentrationdependent manner. From Tilia inflorescences, methanol extract was less efficient than NDGA, ascorbic acid, pyruvate, and DMTU ( < 0.05) but 1.0-fold more efficient than GSH in scavenging 1 O 2 (Table 3), whereas hexane extract was less efficient than NDGA, ascorbic acid, pyruvate, and DMTU ( < 0.05), but 2.0-fold more efficient than GSH in scavenging 1 O 2 (Table 3). In the case of the aqueous extract, it was less efficient than NDGA, ascorbic acid, pyruvate, GSH, and DMTU ( < 0.05) ( Table 4). Regarding Tilia leaves, methanol was less efficient than NDGA, ascorbic acid, pyruvate, and DMTU ( < 0.05) but 9.5-fold more efficient than GSH in scavenging 1 O 2 ( < 0.05) ( Table 4). Hexane extract from leaves was less efficient than NDGA, ascorbic acid, pyruvate, GSH, and DMTU ( < 0.05) (Table 4). Finally, aqueous extract from leaves was unable to scavenge the studied reactive species (data not shown). In accord with the obtained results, the efficiency in scavenging 1 O 2 was in the following order: leaves methanol extract > inflorescences hexane extract > inflorescences methanol extract.

Discussion
In this study, inflorescences and leaves of T. americana, prepared as organic and aqueous extracts, were investigated by analyzing its anticonvulsant and antioxidant properties, as well as the differences in some flavonoids concentrations. Significant differences were found in both activities among the analyzed extracts and flavonoids constituents that depended on the inflorescences or leaves studied. It is the first evidence of the anticonvulsant properties of the inflorescences and leaves of Tilia. In particular, methanol and aqueous extracts were effective against tonic seizure, but methanol extract was more active in generalized seizure or myoclonus; these results suggest more concentrated presence of flavonoids such as quercetin implicate major anticonvulsant response. Nevertheless, the individual evaluation of the flavonoids quercetin, isoquercitrin, and rutin demonstrated that among them exists differential capability to inhibit seizures.
It is known that repeated or single-dose administration of PTZ decreases GABAergic function producing seizures [25]; it is due to its selective blocker action on the chloride ionosphere complex to the -aminobutyric acid A (GABA A ) receptor. It is also known that oxidative stress in the CNS has been shown in several experimental models of epilepsy, such as the PTZ kindling model [26][27][28].
Oxidative stress is defined as an imbalance in the levels of reactive oxygen species (ROS). Generation of ROS is ubiquitous because they are generated during aerobic metabolism, that is, mitochondrial oxidation and other monoamine oxidants. Various defense systems exist in the brain to scavenge ROS, including glutathione peroxidase, vitamin E, vitamin C, and vitamin A [27,29]. In a previous study, pretreatment with hexane or aqueous extracts of the Tilia inflorescences prevented dysfunctional response of the ischemic tissue in a significant manner suggesting a neuroprotector role [30]. The reduced response in ischemic tissues can be prevented by pretreatment with certain agents, such as antioxidants or calcium channels blockers, like verapamil, considered as neuroprotective drugs [31,32]. In this study, we demonstrated that not only the methanol but also a hexane extract from the leaves and flowers contains free radical scavengers that are more concentrated in the methanol extract of the leaves against 1 O 2 . Tilia extracts also display an improved scavenging capacity when compared with the reference compounds suggesting that these possess a neuroprotective activity that might be involved against PTZ-induced seizures due to its antioxidant activity.
Antioxidant and anticonvulsive abilities of natural products have already been demonstrated. In 2010, Aldarmaa et al. [33] showed that ethanol extract of the aerial parts and roots from Astragalus mongholicus trapped hydroxyl radicals (IC 50 root = 4.7 mg/mL; IC 50 aerial parts = 7.8 mg/mL; reference compound = 6.6 mg/mL), diminished lipid peroxidation (IC 50 root = 2.04 mg/mL; IC 50 reference compound = 7.92 mg/mL), and protected against malondialdehydeinduced oxidative damage by ameliorating effects on mitochondrial complexes I and II, malate dehydrogenase, and the mitochondria membrane potential (concentrations from 18 to 72 mg/mL). Moreover, an Astragalus mongholicus extract (150 and 300 mg/kg) diminished malondialdehyde, protein  carbonyl, and ROS levels induced by PTZ in mice [33]. The Astragalus mongholicus extract significantly increased the latency of PTZ-induced seizures and decreased the duration of seizures at doses of 150 and 300 mg/kg [33]. In 2009, Ilhan et al. showed that Nigella sativa oil (12 mL/kg) significantly decreased lipid peroxidation and increased SOD and GPx activity in the brain tissue of PTZ-treated group being more effective at reducing the seizures than valproate (100 mg/kg) [26]. Tilia extracts possess capacity to trap multiple reactive species, including O 2 •− , HO • , HOCl, H 2 O 2, and 1 O 2 . These results suggest that free radial scavenging might be a part of the mechanisms underlying the antinociceptive, anxiolytic, and sedative effects previously observed in experimental models and in human use of traditional medicine for T. americana [7,9,16,18,34]. The methanol extract of leaves showed a significantly higher scavenger activity against 1 O 2 in comparison with the reference compound GSH. It has been demonstrated that antioxidant properties of plants depend on the contain of constituents in leaves, inflorescences, and fruits as well as chemical profile, extract type, part of the plant, species, light exposition, life cycle, and time of plant germination, flowering, and harvest [35][36][37]. Phytochemical studies have reported that flavonoids, such as quercitrin, isoquercitrin, kaempferol, astragalin, hyperoside, tiliroside, quercetin-3,7O-dirhamnoside, and kaempferol-3,7-O-dirhamnoside, are major components of methanol extracts from the Tilia genus [8,18]. The presence of flavonoids in the methanol extract suggests that the anticonvulsant properties shown by Tilia could be due to these compounds, which in previous studies have been involved as responsible anticonvulsants and anxiolytics [16,38]. This is in agreement with the recent study of our group demonstrating that ethyl acetate extract of T. americana var. mexicana possesses high concentration of flavonoids and potential scavenging against 1 O 2 [39].
Based on our present findings, we suggest that the anticonvulsant effects of polar extracts of Tilia may be mediated by reducing oxidative damage in addition to GABAergic and serotoninergic neurotransmission as previously reported. Furthermore, it is important to consider Tilia as an antioxidant that may be a possible therapeutic agent for other neuropathies related to oxidative stress, such as Parkinson's disease [40][41][42][43][44] and Alzheimer's disease [32,[45][46][47].
Tilia species have been used in traditional medicine for their tranquilizing properties by using exclusively the inflorescences (flowers and bracts). It has been observed that during the flowering months (April to June) there is an increase in the marketing of inflorescences of this species, given the belief that the medicinal effect is greater when the infusion of inflorescences is prepared during this period [2]. Our results corroborated that inflorescences of Tilia produce inhibitory activity in the CNS as an anticonvulsive as reported by people using folk medicine. Nevertheless, it is important to mention that differences in the anticonvulsant activity were encountered when Tilia leaves were tested in comparison to the inflorescences. These findings reinforce the medicinal properties of this plant and provide evidence that leaves are not as recommended for this medicinal purpose as it is for inflorescences. The results demonstrated that the presence of flavonoids differs depending on the part of the plant assayed. Major concentrations were observed in the case of quercetin and rutin; however isoquercitrin was more active than these two flavonoids. It has been demonstrated that flavonoids like quercetin, rutin, and kaempferol elicit CNS activity which involves the inhibitory GABAergic system [5,48], but it is 8 Oxidative Medicine and Cellular Longevity the first time to our knowledge that CNS activity is described for isoquercitrin.
The precise mechanisms of action of flavonoids might differ depending on the kind of chemical structure and pharmacological activity, but regarding the antianxiety and sedative-like activities the involvement of the GABAergic and serotonergic systems is the most reported [49][50][51]. In our study, an antioxidant activity was demonstrated for the hexane, methanol, and aqueous crude extracts from inflorescences and leaves and some flavonoids (quercetin, rutin, and isoquercitrin) of Tilia. From these flavonoids, only rutin and isoquercitrin (glycosides of quercetin) were observed to be partial responsible for the anticonvulsant activity of this species suggesting that a synergism among these and likely other constituents are involved in their anticonvulsant activities whose antioxidant properties might also be participating in their neuroprotective effects.
It is recognized that glycosidic moiety increases hydrophilicity influencing pharmacokinetic properties in the body fluids. Glycosylation can strongly influence transport of flavonoids through haematoencephalic barrier modifying the entrance into the brain tissue and its neuropharmacological properties [52]. In particular, the high level of quercetin confers to the extracts an important antioxidant potential, while phenolic compounds are very important plant constituents due to their scavenging abilities associated with their OH groups and to their capacity to chelate transition metal ions [53].
The literature data corroborate the structural features of flavonoids including (i) the ortho dihydroxy structure in the B-ring; (ii) the C2-C3 double bond conjugated with the 4-oxo group; and (iii) the presence of hydroxyl groups in positions 3 and 5. These characteristics are important for the flavonoids free radical scavenging activities [54]. The predominant mechanism of action of catechols is probably via donation of a single electron to the radical cation resulting in the formation of a semiquinone, which can donate a further electron to form the quinine [55].
On the other hand, it has been demonstrated that quercetin and kaempferol flavonoid aglycones isolated from methanol-water extract from T. platyphyllos are stronger scavengers of 1 O 2 in comparison with Trolox (vitamin E hydrophilic derivative) [56]. It has been demonstrated that HO groups located on the B-ring in flavonoids enhance reactivity against O 2 •− , ABTS, and 1 O 2 . [57]. Quercetin with a catechol structure on B-ring (two HO groups in positions 3 and 4 ) showed 4-time higher reactivity towards 1 O 2 in comparison with kaempferol (one HO group in position 4 ) [57]. Additionally, the relevance of hydroxyl group activating the double bond of the C-ring in flavonols in 1 O 2 quenching has been established. In general, flavonol glycosylated forms are weaker antioxidants than their respective aglycones [57]. We would expect more antioxidant activity with quercetin in comparison with isoquercitrin and rutin.
Finally, our results demonstrated the anticonvulsant and antioxidant activities in the inflorescences and leaves of Tilia species that involve the presence of flavonoids. The differences in the pattern and concentrations of these compounds were factors that modify the anticonvulsant properties of Tilia, but quercetin may be useful for the standardization of the extract.

Conclusion
In summary, our results showed that polar extracts of T. americana var. mexicana significantly prevented severity of PTZ-induced seizures and attenuated oxidative stress levels involving the presence of flavonoids such as quercetin, rutin, and isoquercitrin. These extracts and their constituents are part of the anticonvulsant activity that are described in folk medicine.