Evaluation of effects of dichloromethane fraction from Platonia insignis on pilocarpine-induced seizures

Evaluation of effects of dichloromethane fraction from Platonia insignis on pilocarpine-induced seizures

Joaquim S. da Costa Júnior^I; Alexandre de B. F. Ferraz^I; Chistiane Mendes Feitosa^II; Antonia Maria das Graças Lopes Citó^II; Jenifer Saffi^III; Rivelilson Mendes de FreitasIV^*

^ILaboratório de Toxicologia Genética, Programa de Pós-graduação em Toxicologia Genética e Aplicada, Universidade Luterana do Brasil, Brazil

^IIDepartamento de Química, Instituto Federal do Piaui, Brazil

^IIIDepartamento de Ciências Básicas da Saúde, Universidade Federal de Ciências da Saúde de Porto Alegre, Brazil

^IVDepartamento de Bioquímica e Farmacologia, Instituto Federal do Piaui, Brazil

ABSTRACT

The objective of present study was to evaluate the antioxidant and anticonvulsant activities of dichloromethane fraction (DMF) from Platonia insignis Mart., Clusiaceae. The DMF from P. insignis (2 mg/kg) was tested by intraperitoneal (i.p.) to evaluate effects on lipid peroxidation level, nitrite formation, as well as on locomotor and anticonvulsant activities. Wistar rats were treated with, (saline/Tween 80 0.5%, i.p., control group), DMF (2 mg/kg, i.p., DMF group), pilocarpine (400 mg/kg, i.p., P400 group), or the combination of DMF (2 mg/kg, i.p.) and pilocarpine (400 mg/kg, i.p., DMF plus P400). After the treatments all groups were observed for 24 h. In P400 group rats there was a decrease in the motor activity when compared with control group. In DMF plus P400 co-administered rats was observed an increase in motor activity when compared with P400 group. In P400 group rats there was a significant increase in lipid peroxidation and nitrite levels. In DMF plus P400 co-administered rats, antioxidant treatment significantly reduced the lipid peroxidation level and nitrite content after seizures. Previous findings strongly support the hypothesis that oxidative stress occurs in rat striatum during pilocarpine-induced seizures, and our results imply that strong neuprotective effect on this brain region could be achieved using DMF from P. insignis.

Keywords: lipid peroxidation, nitrite, pilocarpine, P. insignis, seizures

Introduction

Medicinal plants play a key role not only in self-medication but also in medical practice of the wild world. The power of the chemical and biochemical techniques in studying such plants has led to discovery of important agents for treatment of long-last diseases. These discoveries emphasize the pharmaceutical potential of the medicinal plants (Bezerra et al., 2008).

The term epilepsy is collectively designated for a group of chronic central nervous system disorders characterized by spontaneous occurrence of seizures generally associated with the loss of consciousness and body movements of convulsions (Chauhan et al., 1988). The search for antiepileptic compounds with more selective activity and lower toxicity continues to be an area of intensive investigation in medicinal chemistry (Malawska, 2005). Various phytochemical and pharmacological studies have been carried out on these anticonvulsant plants (Quintans Júnior et al., 2008a).

Platonia insignis Mart., Clusiaceae, commonly known as "bacuri", is a thick-skinned fruit, with approximate dimension of an orange, which contains a large quantity of resins. The pulp enclosing the seeds is white, bittersweet, with a pleasant smell and taste. The fruit can be consumed raw or in the form of juice, ice-cream or jam (Alves & Jennings, 1979). A number of xanthone have been isolated from plants belonging to this family (Ollis et al., 1965).

Many new polyisoprenylated benzophenones with a bicycle-[3.3.1]-nonane-2,4,9-trione core structure have been isolated from plants in the Clusiaceae family, and their potent biological properties have been the subject of several studies. This review summarizes the biological activities reported for these secondary metabolites including cytotoxic, antimicrobial, antioxidant, and anti-inflammatory activities (Acuna et al., 2009; Costa Júnior et al., 2011). The polycyclic polyprenylated acylphloroglucinols exhibit a wide variety of biological activities such as antimicrobial, antidepressant, antioxidant, cytotoxic, and antiviral activities (Ciochina & Grossman, 2006).

The fats form the P. insignis seeds (bacuri fat) are yellowish solid rich in triacylglycerols and fatty acid (Bentes et al., 1986). The composition of bacuri fat, the seed fat of P. insignis and found that its chief component acids are palmitic and oleic acid, with smaller proportions of stearic and palmitoleic acids and probably traces of myristic, arachidic, and linoleic acids.

Omega-3 and omega-6 polyunsaturated fatty acids (PUFA) are dietary fatty acids that are involved in a myriad of physiologic processes in the brain. There is some evidence suggesting that PUFA and particularly omega-3 PUFA may have anticonvulsant effects, both in humans and animals (Taha et al., 2010).

Natural xanthones have been reported in genera Calophyllum, Platonia, Symphonia, and Kielmeyera (Jackson et al., 1966). A series of xanthone derivatives have shown pronounced anticonvulsant activity (Marona et al., 2001; Marona et al., 2008). The aim of present study was to examine the effects of DMF from P. insignis on lipid peroxidation level and nitrite formation in rat striatum, as well as research their anticonvulsant activity in adult rats prior to pilocarpine-induced seizures.

Materials and methods

Drugs

The drugs used pilocarpine hydrochloride, trichloroacetic acid, thiobarbituric acid, sodium nitrite, and polyoxyethylene-sorbitan monolated (Tween 80) were purchased from Sigma (USA). Agents were administrated by intraperitoneally (i.p.) route at a dose volume of 0.1 mL/10 g.

Plant material and preparation of DMF from P. insignis

The Platonia insignis Mart., Clusiaceae, fruits were collected at Barras, Piauí State, Brazil, in March 2009. A voucher specimen has been identified and deposited at the "Graziela Barroso", Herbarium of Biology Department of Federal University of Piauí, Brazil (Voucher No.: ICN TEPB 27.164). The seeds collected from the fruits of P. insignis were dried at 55 °C under shade and powdered mechanically. Crush yielded of seeds (848 g) was extracted with n-hexane (63%, w/w), followed by 95% ethanol (5.8%, w/w) in a Soxhlet apparatus (8 h for each solvent). In the ethanol extract it was added 100 mL of water, which was then fractionated using polarity increasing solvents. The ethanol extract was fractionated with dichloromethane (8 x 100 mL) to obtain a dichloromethane soluble fraction. The fraction was concentrated in a vacuum evaporator. The concentrated extract was finally freeze-dried to get the yield of 3.4% of DMF. The dried extract was kept at 4 °C in refrigerator in the air tight bottles until use.

Animals and treatment protocols

Adult male Wistar rats (250-280 g) were maintained in a temperature controlled room (26±1 °C) with a 12-h light/dark cycle and food and water ad libitum (Nutrilabor, Campinas, Brazil). All experiments were performed according to the guide for the care and use of laboratory the US Department of Health and Human Services, Washington, DC (1985).

A total of 96 rats were treated with either 2 mg/kg DMF from P. insignis (i.p., DMF) or vehicle (saline/Tween 80 0.5%, i.p.), and 30 min later the treatments 24 rats from each above group were randomized to pilocarpine hybrochloride administration (400 mg/kg, i.p., P400). Thus there are four groups of rats in this set of experiments: group 1, DMF and P400 co-administration (n=24); group 2, P400 plus vehicle treatment (n=24); group 3, DMF alone administration (n=24); and group 4, vehicle treatment serves as control (n=24). After the treatments, the animals were recorded in 30 cm x 30 cm chambers with: latency to first seizure (any one of the behavioral indices typically observed after pilocarpine administration: wild running, clonuses, tonus, clonic-tonic seizures) and number of animals that died after pilocarpine administration. Previous work has shown that convulsions and deaths occurred within 1 and 24 h respectively post pilocarpine injection, so we decided to record the phenotypes of the animals for 24 h after pilocarpine administration (Turski et al., 1983). At the end of observations, the survivors were killed by decapitation and their brains were dissected on ice to remove striatum for determinations of lipid peroxidation level and nitrite content. The pilocarpine group (P400) was constituted by those presented seizures and status epilepticus (SE) (Freitas et al., 2005).

The drug dosages of pilocarpine (400 mg/kg) and DMF (2 mg/kg) were determined by previous study in our lab (Barros et al., 2007; Xavier et al., 2007) and the present study (data not shown). The drug doses used in this present study are not equivalent to those used by humans because rats have different metabolic rates.

Behavioral effects and locomotor activity

Behavioral screening of the rats was performed following parameters described by Almeida et al. (1999) and animals were observed at 24 h after i.p. administration of DMF of P. insignis (2 mg/kg, i.p.). During 24 h were observed the occurrence of the following general signs (piloerection, prostration, writhing, evacuation, grooming, dyspnea, sedation, analgesia and palpebral ptosis).

Rats were divided into four groups of seven animals each. Vehicle received saline/Tween 80 0.5% (control group) and the tested groups were administered with DMF (2 mg/kg, i.p.). The spontaneous locomotor activity of the animals was assessed in a cage activity (50 cm × 50 cm × 50 cm) after 24 h of treatment (Asakura et al., 1993).

Determinations of lipid peroxidation level in striatum of adult rats pretreated with DMF from P. insignis prior to pilocarpine-induced seizures

For all experimental procedures, 10% (w/v) homogenates of the area of the brain investigated were prepared for all groups. Lipid peroxidation levels in the DMF plus P400 group (n=6), P400 group (n=6), DMF group (n=6) and control animal (n=9) were analyzed by measuring the thiobarbituric-acid-reacting substances in homogenates (Draper & Hadley, 1990). Briefly, the homogenates were mixed with 1 mL 10% trichloroacetic acid and 1 mL 0.67% thiobarbituric acid, and were heated in a boiling water bath for 15 min, and then butanol (2:1, v/v) was added to the solution. After centrifugation (800 x g, 5 min), TBARS determinations were performed spectrophotometrically at 535 nm and expressed as nmol of malondialdehyde (MDA)/g wet tissue.

Determinations of nitrite content in striatum of adult rats pretreated with DMF from P. insignis prior to pilocarpine-induced seizures

To determine nitrite contents of control group (n=9), DMF plus P400 group (n=6), P400 group (n=6) and DMF group (n=6), the 10% (w/v) homogenates were centrifuged (800 x g, 10 min). The supernatants were collected, and nitric oxide production was determined based on the Griess reaction (Green et al., 1981). Briefly, 100 μL supernatant was incubated with 100 μL of the Griess reagent at room temperature for 10 min. Nitrite concentration was determined from a standard nitrite curve generated using NaNO₂. Nitrite determinations were performed spectrophotometrically at 550 nm using a microplate reader and expressed as nM.

Statistical analysis

Results of latency to first seizure, locomotor activity and neurochemical alterations were compared by one-way analysis of variance (ANOVA) followed by Student-Newman-Keuls test (p<0.05) (Graphpad program Intuitive, Software for Science, San Diego, CA). The number of animals that seized and the number that survived were calculated as percentages (seizures percentage and survival percentage, respectively), and compared with a nonparametric test (χ²).

Results

Behavioral alterations after pretreatment with DMF from P. insignis prior to pilocarpine-induced seizures

Pilocarpine induced the first seizure at 35.00±0.70 min. All the animals studied showed generalized tonic-clonic convulsions with SE, and 30% survived the seizures. All animals pretreated with DMF from P. insignis were observed for 24 h before pilocarpine injection and their manifested alterations in behavior, such as peripheral cholinergic signs (100%), tremors (50%), staring spells, facial automatisms, wet dog shakes, rearing and motor seizures (100%) developed progressively within 1-2 h into a long-lasting SE (100%). Table 1 shows that DMF (2 mg/kg) administration before pilocarpine treatment reduced by 70% the percentage of animals that seized (p<0.0001), increased latency (341%) to the first seizure (154.21±1.54 min) (p<0.0001) and increased (50%) the survival (p<0.0001), when compared to the pilocarpine only group. None of the control animals (vehicle or dichloromethane fraction) showed seizures (Table 1).

Locomotor activity after pretreatment with DMF from P. insignis prior to pilocarpine-induced seizures

In P400 group was observed significant decreases of 9 and 21% of number of crossings and rearing, when compared to vehicle group, respectively. In dose of 2 mg/kg of DMF caused significant increase of 11 and 7% of ambulation and rearing, when compared to P400 group, respectively. In addition, the pretreatment with DMF, 30 min before administration of pilocarpine increased number of crossings (48%, p<0.001) and rearing (49%, p<0.001) when compared to the P400 group (Figure 1). On the other hand, none of the control animals (vehicle or dichloromethane fraction) showed alterations in number of crossings and rearing (Figure 1).

Lipid peroxidation level and nitrite content in striatum of adult rats pretreated with DMF from P. insignis prior to pilocarpine-induced seizures

Effects of DMF from P. insignis in lipid peroxidation and nitrite concentrations during seizures induced by pilocarpine are presented in Figure 2. Lipid peroxidation was markedly increased of in pilocarpine group in comparison with the corresponding values of the vehicle group. During acute phase of seizures induced by pilocarpine a significant increase (89%) in thiobarbituric-acid-reacting substances (p<0.0001) was observed. Seizures induced by pilocarpine produced a significant increase in striatal nitrite content (94%, p<0.0001, Figure. 2). Rats pretreated with DMF showed decrease in lipid peroxidation level (48%, p<0.001) and nitrite content (49%, p<0.001) when to compared with the pilocarpine group (Figure 2). In addition, the pretreatment with DMF, 30 min before administration of pilocarpine also reduced lipid peroxidation level (94%, p<0.0001) and nitrite content (51%, p<0.05) when compared to the vehicle group (Figure 2). On the other hand, none of the control animals (vehicle or dichloromethane fraction) showed changes in lipid peroxidation level and nitrite content (Figure 2).

Discussion

In folk medicine of the Brazilian Northeast P. insignis seed oil's is used for treatment of eczemas, herpes, and diarrheas (Agra et al., 2007). In pharmacological behavioral screening, the animals treated with DMF of P. insignis showed increase of response to touches and increasing of motor activity. These data are indicative of stimulatory activity of the CNS (Almeida et al., 1999).

The possible CNS antioxidant and anticonvulsant activities of ethanol extract (Costa Júnior et al., 2010) and garcinielliptone FC (Costa Júnior et al., 2011) from P. insignis were investigated in vitro assays and in animal models. In present study we investigated effects of DMF from P. insignis in animal models. The mice treated with DMF presented behavioral alterations, such increased ambulation, palpebral ptosis, and stimulation. These behavioral changes suggest a possible effect on CNS; however, they are different to drugs that reduce the CNS activity (Morais et al., 2004; Netto et al., 2009).

Previous studies using low doses of ethanol extract and fractions from P. insignis demonstrated pharmacological activity in central nervous system, our experiments only with one dose of DMF present results anticonvulsant and antioxidant for this fraction. However, we need to investigate the same dose of DMF, as has central activity in other tests in vitro and in animal models of seizures (pentylenetetrazol and picrotoxin) to clarify the possible action mechanism of this fraction.

Dichloromethane fraction of P. insignis at the highest dose caused a significant increase of ambulation in the test of spontaneous movement after 24 h in the dose of 2 mg/kg, corroborating with the hypothesis that DMF of P. insignis did not reduce the CNS activity. Our data suggest that DMF may be a stimulatory to the CNS, since studies shows that reduction of the ambulation of the animals is characteristic of depressant drugs (Carlini, 2003; Freire et al., 2006; Leite et al., 2008; Quintans Júnior et al., 2008b).

The increase of the locomotor activity was observed and it can be due to either through a stimulatory effect of the DMF of P. insignis on CNS or by absence of muscular relaxant activity in the periphery system. Our results indicate that DMF could possess a stimulator activity.

The molecular observations of epilepsy include the temporal correlation between free radical generation and the development of seizures in some pathological conditions, and the protective efficacy of antioxidant treatments against some types of seizures. P. insignis, one of the effective antioxidant, not only has antioxidant functions, but also has functions in pro-oxidant (Hosni et al., 2010; Lenta et al., 2007). Previous studies indicated that P. insignis has antioxidant activity in several animal models (Wu et al., 2005; Wu et al., 2008; Iinuma et al., 1996; Gustafson et al., 1992). The effects of DMF of P. insignis leaves in CNS have not yet been determined, therefore, it would be important to conduct these studies to clarify its brain action mechanism in pilocarpine-induced seizures. In this study, we demonstrated a role of DMF from P. insignis against lipid peroxidation and nitrite formation produced by pilocarpine-induced seizures.

In the present study we investigated the influence of DMF from P. insignis on the level of lipid peroxidation and nitrite content in the rat striatum during pilocarpine-induced seizures. Generation of reactive oxygen species is currently viewed as one of the process through which epileptic activity exert their deleterious effects on brain (Rauca et al., 2004). These reactive oxygen species in the absence of an efficient defense mechanism cause peroxidation of membrane polyunsaturated fatty acids (Castagne et al., 1999). Brain is particularly susceptible to peroxidation due to simultaneous presence of high levels of polyunsaturated fatty acids and iron (Halliwell & Gutteridge, 1999) which are the targets of free radical damage (Gottlieb et al., 2006; Halliwell & Gutteridge, 1989). We showed the lipid peroxidation was rising in striatum homogenate of rats after 24 h of acute phase of seizures. The increase of lipid peroxidation was reflected by the rise of thiobarbituric-acid-reacting substances level which may be related to its intermediate free radicals formed during pilocarpine-induced seizures.

Literature has shown that pilocarpine-induced seizures led to changes in nitric oxide metabolism, and increased the production of its metabolites (nitrite and nitrate). The increased metabolites may interact with glutamatergic receptors to produce part of its stimulatory action on the central nervous system (Maczurek et al., 2008; Michiels et al., 1994). The reduction in nitrite content, after pretreatment with DMF from P. insignis, is most readily explained as a consequences of radical formation inhibiting, scavenges reactive oxygen species and lipid peroxidation products (Tejada et al., 2006).

Herein, we clearly showed that DMF from P. insignis decreased the frequency of pilocarpine-induced seizures and increased the survival rate. In our knowledge, these effects of DMF on lipid peroxidation and nitrite formation observed during acute phases of pilocarpine-induced seizures have not been reported before. Thus, these findings might have important implications for understanding the mechanism of epilepsy to promote new advances in the development of selective and targeted antiepileptic drugs. DMF from P. insignis might protect the striatum against neuronal damages regularly observed during seizures.

Our results confirm data previously reported in the literature that demonstrate anticonvulsant activity of ethanol extract of Hypericum perforatum in mice belonging to the same family of plant evaluated in this study (Hosseinzadeh et al., 2005; Vyawahare et al., 2007). Further investigations of effects of DMF from P. insignis against necrosis, apoptosis and/or autophagy observed during the acute phase of this epilepsy model are in progress to confirm its neuroprotective effects.

Acknowledgements

The authors are grateful to Fundação de Amparo a Pesquisa do Estado do Piauí, Instituto Federal do Piauí, Conselho Nacional de Desenvolvimento Científico e Tecnológico.

Received 9 Nov 2010

Accepted 8 Jun 2011

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Publication Dates

• Publication in this collection
  09 Sept 2011
• Date of issue
  Dec 2011

History

• Received
  09 Nov 2010
• Accepted
  08 June 2011